Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof

ABSTRACT

The present disclosure provides a humanized anti-CD37 small modular immunopharmaceutical (SMIP) molecule, as well as synergistic combination therapies of CD37-specific binding molecules (such as anti-CD37 SMIP proteins or antibodies) with bifunctional chemotherapeutics (such as bendamustine) that can be administered concurrently or sequentially, for use in treating or preventing B cell related autoimmune, inflammatory, or hyperproliferative diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/190,067 filed Apr. 11, 2008, wherethis provisional application is incorporated herein by reference in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 910180_(—)420PC_SEQUENCE LISTING.txt. The textfile is 286 KB, was created on April 10, 2009, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND

1. Technical Field

The present disclosure generally provides compositions and methods fortreating B-cell disorders and, more specifically, a humanized anti-CD37small modular immunopharmaceutical (SMIP) molecule, as well assynergistic combination therapies of CD37-specific binding moleculeswith bifunctional chemotherapeutics for use in treating or preventingB-cell related autoimmune, inflammatory, or hyperproliferative diseases.

2. Description of the Related Art

The human immune system generally protects the body from invadingforeign substances and pathogens. One component of the immune system isB lymphocytes, also referred to as B-cells, which produce antibodiesthat protect the body by binding to, and in some cases mediatingdestruction of, a foreign substance or pathogen. In some instances,however, the immune system functions can go awry and disease results.For example, there are numerous cancers, autoimmune diseases, andinflammatory diseases that involve uncontrolled proliferation ofB-cells.

B-cells can be identified by molecules on their cell surface, such asCD37. CD37 is a heavily glycosylated 40-52 kDa protein that belongs tothe tetraspanin transmembrane family of cell surface antigens, which ishighly expressed on normal antibody-producing B-cells but not onpre-B-cells or plasma cells. In addition to normal B-cells, almost allmalignancies of B-cell origin are positive for CD37 expression,including chronic lymphocytic leukemia (CLL), non-Hodgkins lymphoma(NHL), and hairy cell leukemia (Moore et al., J. Pathol. 152:13 (1987);Merson and Brochier, Immunol. Lett. 19:269 (1988); and Faure et al., Am.J. Dermatopathol. 12:122 (1990)).

A few CD37 specific immunotherapies have been developed. An IgG1 murinemonoclonal antibody specific for CD37, MB-1, was labeled with ¹³¹I andtested in a clinical trial in the treatment of NHL (see Press et al., J.Clin. Oncol. 7:1027 (1989); Bernstein et al., Cancer Res. (Suppl.)50:1017 (1990); Press et al., Front. Radiat. Ther. Oncol. 24:204 (1990);Press et al., Adv. Exp. Med. Biol. 303:91 (1991) and Brown et al., Nucl.Med. Biol. 24:657 (1997)). The MB-1 antibody lacks Fc effectorfunctions, such as antibody-dependent cellular cytotoxicity (ADCC), andthe naked MB-1 antibody did not inhibit tumor growth in an in vivoxenograft model (Buchsbaum et al., Cancer Res. 52:6476 (1992)). Inaddition, an immunoconjugate having adriamycin linked to G28-1, anothermurine monoclonal anti-CD37, was administered to mice and shown to beinternalized with adriamycin being released intracellularly (see,Braslawsky et al., Cancer Immunol. Immunother. 33:367 (1991)). Anengineered fusion protein, termed a small modular immunopharmaceutical(SMIP™) product, directed to CD37 is currently being tested in humans(see, e.g., US Patent Application Publications 2003/0133939 and2007/0059306).

Although there has been extensive research carried out on antibody-basedtherapies, there remains a need in the art for alternative or improvedcompositions and methods for treating B-cell associated disorders ordiseases.

BRIEF SUMMARY

In one aspect, the present disclosure provides humanized CD37-specificbinding molecules and a method for reducing B-cells or treating adisease associated with aberrant B-cell activity comprisingadministering to a subject in need thereof an effective amount of ahumanized CD37-specific binding molecule provided herein.

In certain embodiments, the present disclosure provides a humanizedCD37-specific binding molecule, comprising from amino terminus tocarboxyl terminus: (i) a humanized heavy chain variable region, (ii) alinker as set forth in SEQ ID NO:229, (iii) a humanized light chainvariable region, (iv) an IgG1 hinge, (v) human IgG1 CH2 region, and (vi)human IgG1 CH3 region, wherein (a) the humanized heavy chain variableregion comprises from amino terminus to carboxyl terminus: a humanizedheavy chain FR1, a heavy chain CDR1 as set forth in SEQ ID NO:63, ahumanized heavy chain FR2, a heavy chain CDR2 as set forth in SEQ IDNO:65, a humanized heavy chain FR3, a heavy chain CDR3 as set forth inSEQ ID NO:67, 68 or 69, and a humanized heavy chain FR4, and (b) thehumanized light chain variable region comprises from amino terminus tocarboxyl terminus: a humanized light chain FR1, a light chain CDR1 asset forth in SEQ ID NO:61 or 62, a humanized light chain FR2, a lightchain CDR2 as set forth in SEQ ID NO:64, a humanized light chain FR3,and a light chain CDR3 as set forth in SEQ ID NO:66, and a humanizedlight chain FR4.

In certain embodiments of the above humanized CD37-specific bindingmolecules, the humanized heavy chain FR1 comprises SEQ ID NO:144, thehumanized heavy chain FR2 comprises SEQ ID NO:151, the heavy chain FR3comprises SEQ ID NO:158, and the heavy chain FR4 comprises SEQ ID NO:161or 162.

In certain embodiments of any one of the above humanized CD37-specificbinding molecules, the humanized light chain FR1 comprises SEQ IDNO:171, the light chain FR2 comprises SEQ ID NO:182, the light chain FR3comprises SEQ ID NO:195, and the light chain FR4 comprises SEQ IDNO:206.

In a related aspect, the present disclosure provides a CD37-specificbinding molecule that comprises the amino acid sequence as set forth inSEQ ID NO:253.

In certain embodiments, the CD37-specific binding molecule consistsessentially of the amino acid sequence as set forth in SEQ ID NO:253.

In certain embodiments, the CD37-specific binding molecule consists ofthe amino acid sequence as set forth in SEQ ID NO:253.

In a related aspect, the present disclosure also provides an isolatednucleic acid molecule that comprises a nucleotide sequence encoding ahumanized CD37-specific binding molecule provided herein.

In another related aspect, the present disclosure provides a vector thatcomprises an isolated nucleic acid molecule that encodes a humanizedCD37-specific binding molecule provided herein.

In another related aspect, the present disclosure provides a host cellthat comprises the above-described vector.

The present disclosure also provides a composition that comprises ahumanized CD37-specific binding molecule provided herein and apharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method for reducingB-cells or treating a disease associated with aberrant B-cell activity,comprising administering to a subject in need thereof an effectiveamount of a humanized CD37-specific binding molecule provided herein.

In certain embodiments, the disease associated with aberrant B-cellactivity is a B-cell lymphoma, a B-cell leukemia, a B-cell myeloma, adisease characterized by autoantibody production, or a diseasecharacterized by inappropriate T-cell stimulation associated with aB-cell pathway.

In certain embodiments, the disease characterized by autoantibodyproduction is idiopathic inflammatory myopathy, rheumatoid arthritis,myasthenia gravis, Grave's disease, type I diabetes mellitus, multiplesclerosis, an autoimmune disease, dermatomyositis, polymyositis, orWaldenstrom's macroglobinemia.

In certain embodiments, the disease associated with aberrant B-cellactivity is chronic lymphocytic leukemia (CLL).

In another aspect, the present disclosure provides compositions andmethods for the combined use of CD37-specific binding molecules andbifunctional chemotherapeutics to reduce B-cells or treat a diseaseassociated with aberrant B-cell activity. A surprising result of thiscombination is that these compounds act synergistically, which resultsin an increased B-cell reduction.

For example, the present disclosure provides a composition thatcomprises a CD37-specific binding molecule and bendamustine. In certainembodiments, the CD37-specific binding molecule is a

CD37-specific antibody or SMIP, such as a humanized antibody orhumanized SMIP.

In certain embodiments, the CD37-specific binding molecule competes withG28-1 mAb in CD37-specific binding.

In certain embodiments, the CD37-specific binding molecule is ahumanized CD37-specific binding molecule provided herein, such as ahumanized CD37-specific binding molecule that comprises, consistsessentially of, or consists of, the amino acid sequence as set forth inSEQ ID NO:253.

In a related aspect, the present disclosure provides a method forreducing B-cells or treating a disease associated with aberrant B-cellactivity, comprising administering to a subject in need thereof aneffective amount of a CD37-specific binding molecule and bendamustine.

In certain embodiments, the disease associated with aberrant B-cellactivity is a B-cell lymphoma, a B-cell leukemia, a B-cell myeloma, adisease characterized by autoantibody production, or a diseasecharacterized by inappropriate T-cell stimulation associated with aB-cell pathway.

In certain further embodiments, the disease characterized byautoantibody production is idiopathic inflammatory myopathy, rheumatoidarthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus,multiple sclerosis, an autoimmune disease, dermatomyositis,polymyositis, or Waldenstrom's macroglobinemia.

In certain other embodiments, the disease associated with aberrantB-cell activity is chronic lymphocytic leukemia (CLL).

In certain embodiments, the CD37-specific binding molecule andbendamustine are administered concurrently.

In certain other embodiments, the CD37-specific binding molecule andbendamustine are administered sequentially.

In certain embodiments, the CD37-specific binding molecule andbendamustine are formulated together.

In certain embodiments, the CD37-specific binding molecule is aCD37-specific antibody or SMIP, such as a humanized antibody or ahumanized SMIP.

In certain embodiments, the CD37-specific binding molecule competes withG28-1 mAb in CD37-specific binding.

In certain embodiments, the CD37-specific binding molecule is ahumanized CD37-specific binding molecule provided herein, such as ahumanized CD37-specific binding molecule that comprises, consistsessentially of, or consists of, the amino acid sequence as set forth inSEQ ID NO:253.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows heavy and light chain variable region amino acid sequencealignments of mouse G28.1 and CAS-024 sequences, along with a consensusidentity sequence.

FIGS. 2A-2S show the size exclusion chromatography (SEC) chromatogramsof CAS-001, CAS-002, CAS-003, and CAS-024. The peaks of interest (POI)have 98-99% of the SMIP molecules being purified. CAS-024 has a verysharp and symmetrical peak (indicating homogeneity), whereas CAS-001,CAS-002, and CAS-003 peaks have a slight shoulder (where uponintegration, the shoulder accounts for about 35% of the POI), whichindicates a heterogenous population of molecules.

FIG. 3 is graph showing how various anti-CD37 specific SMIP proteinscompete with the parent CAS-006 molecule (chimeric anti-CD37 SMIPprotein, mVLmVH) for binding to CD37 on Ramos cells, which provides anindication on the affinity of binding as compared to the parentmolecule. CAS-024 (hVHhVL) has substantially the same affinity for CD37as does CAS-006, whereas the other molecules (CAS-001, CAS-002, andCAS-003, all hVLhVH) have a 2- to 4-fold decrease in affinity.

FIGS. 4A and 4B are graphs of additional binding competition assaysagainst CAS-006 (labeled as SMIP-016 in these graphs). Here, mouse-humanhybrid SMIP molecules (CAS-014 mVHhVL and CAS-017 hVLmVH) have anaffinity that is higher than CAS-006, whereas CAS-024 shows the samebinding affinity as CAS-006 and CAS-003 (hVLhVH) has a lower bindingaffinity.

FIGS. 5A-5E show competitive binding between various different anti-CD37antibodies and CAS-006 (a chimeric anti-CD37 SMIP molecule).

FIGS. 6A and 6B show that CAS-024 was statistically superior to Rituxan®in the in vivo treatment of an animal model of follicular lymphoma asshown by (A) survival rate and (B) tumor-free percentage.

FIG. 7 shows that CAS-024 acts synergystically with chemotherapeuticagents fludarabine and vincristine to kill mantle cell lymphoma (MCL)cells, Rec-1 cells.

FIG. 8 is a bar graph showing the level of depletion of peripheral bloodlymphocytes in human patients treated with an anti-CD37 SMIP molecule ofthis disclosure.

FIG. 9 shows the lymphocyte depletion and course of treatment forpatient BJB. BJB (part of Cohort 7) was treated with 3.0 mg/kg on days1, 3 and 5 the first week followed by 3 weekly doses in the first cycle,and this same treatment was administered in a second cycle. Patient BJBshowed a dramatic drop in lymphocytes (within 48 hrs), showed a decreasein palpable lymph nodes by day 4, and continues to respond to treatment.

FIG. 10 shows the lymphocyte depletion and course of treatment forpatient GRP. GRP (part of Cohort 4) was treated with 1.0 mg/kg once aweek for four weeks as the first cycle, and then two months later wastreated in the same way in a second cycle. Patient GRP showed a dramaticdrop in lymphocytes (within 2 weeks), showed a 36% decrease in lymphnode size by CT scan, a decrease in spleen size, improved hemoglobinlevel, and continues to respond to treatment.

FIG. 11 shows a combination index (CI) plot for inhibitory effects ofCAS-024 and bendamustine against Rec-1 cell growth.

FIG. 12 shows inhibitory effects of chlorambucil alone and incombination with CAS-024 on SU-DHL-6 cell growth.

FIG. 13 shows a combination index plot for inhibitory effects of CAS-024and chlorambucil on SU-DHL-6 cell growth.

FIG. 14A shows tumor volume comparisons in tumor-bearing mice resultedfrom injections of DOHH2 cells and subsequently treated with huIgG(Human IgG, R&D Systems), CAS-024, bendamustine, and the combination ofCAS-024 and bendamustine. FIG. 14B shows tumor volume of individual miceon day 13 relative to day 0.

FIG. 15 shows mean tumor volumes over time in tumor-bearing miceresulted from injections of DOHH2 cells and subsequently treated withhuIgG, CAS-024, bendamustine, and the combination of CAS-024 andbendamustine. Values are the mean±the standard error of the mean foreach measurement day. Curves for each group end after one or more of themice in the group were euthanized.

FIG. 16 shows survival percentages over time of tumor-bearing miceresulted from injections of DOHH2 cells and subsequently treated withhuIgG, CAS-024, bendamustine, and the combination of CAS-024 andbendamustine.

FIG. 17 shows incidence of tumor free mice over time after treatmentswith huIgG, CAS-024, bendamustine, and the combination of CAS-024 andbendamustine.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides the CD37-specific bindingmolecule CAS-024 (SEQ ID NO:253), which is a humanized version ofCAS-006 (a small modular immunopharmaceutical (SMIP) protein having theimmunoglobulin variable regions from mouse anti-human CD37 monoclonalantibody G28-1). The CAS-024 SMIP protein is unexpectedly (1) expressedat up to about 25-fold higher levels than other humanized versions ofCAS-006 (such as CAS-002, CAS-003; see Examples 2 and 5), (2) capable ofbinding CD37 as well as CAS-006 while other humanized versions do not(see Examples 4 and 5), and (3) produced as a homogenous population ofmolecules as compared the heterogenous nature of other humanizedversions (see Example 3). Additionally, the instant disclosure providesthe CD37-specific binding molecule CAS-024 (SEQ ID NO:253) for use inmethods for reducing B-cells or treating disease associated withaberrant B-cell activity comprising administering to a subject in needthereof an effective amount of CAS-024 provided herein.

In another aspect, the present disclosure provides compositions andmethods for the combined use of any CD37-specific binding molecule andbifunctional chemotherapeutics (such as bendamustine) to reduce B-cellsor treat a disease associated with aberrant B-cell activity. Asurprising result of this combination is that this combination ofcompounds acts synergistically and results in a substantially moreeffective therapeutic regimen.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof mean ±20% of the indicated range, value, or structure, unlessotherwise indicated. It should be understood that the terms “a” and “an”as used herein refer to “one or more” of the enumerated components. Theuse of the alternative (e.g., “or”) should be understood to mean eitherone, both, or any combination thereof of the alternatives. As usedherein, the terms “include” and “comprise” are used synonymously. Inaddition, it should be understood that the individual compounds, orgroups of compounds, derived from the various combinations of thestructures and substituents described herein, are disclosed by thepresent application to the same extent as if each compound or group ofcompounds was set forth individually. Thus, selection of particularstructures or particular substituents is within the scope of the presentdisclosure.

A “binding domain” or “binding region” according to the presentdisclosure may be, for example, any protein, polypeptide, oligopeptide,or peptide that possesses the ability to specifically recognize and bindto a biological molecule (e.g., CD37) or complex of more than one of thesame or different molecule or assembly or aggregate, whether stable ortransient. A binding region includes any naturally occurring, synthetic,semi-synthetic, or recombinantly produced binding partner for abiological molecule or other target of interest. A variety of assays areknown for identifying binding domains of the present disclosure thatspecifically bind a particular target, including Western blot, ELISA, orBiacore analysis.

Binding domains and fusion proteins thereof of this disclosure can becapable of binding to a desired degree, including “specifically orselectively binding” a target while not significantly binding othercomponents present in a test sample, if they bind a target molecule withan affinity or K_(a) (i.e., an equilibrium association constant of aparticular binding interaction with units of 1/M) of, for example,greater than or equal to about 10⁵ M⁻¹, 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸M³¹ ¹, 10⁹M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M³¹ ¹, 10¹² M⁻¹. “High affinity” binding domainsrefers to those binding domains with a K_(a) of at least 10⁷ M⁻¹, atleast 10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹ M⁻¹ ,at least 10¹² M⁻¹, at least 10¹³ M⁻¹, or greater. “Low affinity” bindingdomains refers to those binding domains with a K_(a) of up to 5×10⁷ M⁻¹,up to 10⁷ M⁻¹, up to 10⁶ M⁻¹, up to 10⁵ M⁻¹, or less. Alternatively,affinity may be defined as an equilibrium dissociation constant (K_(d))of a particular binding interaction with units of M (e.g., 10⁻⁵ M to10⁻¹³ M). Affinities of binding domain polypeptides and fusion proteinsaccording to the present disclosure can be readily determined usingconventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y.Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or theequivalent).

The term “CD37-specific binding molecules” refer to a protein,polypeptide, oligopeptide or peptide that specifically binds to CD37with a Ka of at least about 10⁶ M⁻¹ (e.g., at least about 10⁷ M⁻¹, 10⁸M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹).

The term “CD37-specific binding domain” refers to a portion or a domainof a CD37-specific binding molecule responsible for the specific CD37binding of the molecule. A CD37-specific binding domain itself (i.e.,without any other portion of the CD37-specific binding molecule) bindsto CD37 with a Ka of at least about 10⁶ M⁻¹ (e.g., at least about 10⁷M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹). ACD37-specific binding domain itself may be sufficient as a CD37-specificbinding molecule. Exemplary CD37-specific binding domains includeCD37-specific scFv and Fab fragments, which can be derived fromanti-CD37 antibodies, such as monoclonal antibody G28-1.

Terms understood by those in the art as referring to antibody technologyare each given the meaning acquired in the art, unless expressly definedherein. For example, the terms “V_(L)” and “V_(H)” refer to the variablebinding region derived from an antibody light and heavy chain,respectively. The variable binding regions are made up of discrete,well-defined sub-regions known as “complementarity determining regions”(CDRs) and “framework regions” (FRs). The terms “C_(L)” and “C_(H)”refer to an “immunoglobulin constant region,” i.e., a constant regionderived from an antibody light or heavy chain, respectively, with thelatter region understood to be further divisible into C_(H1), C_(H2),C_(H3) and C_(H4) constant region domains, depending on the antibodyisotype (IgA, IgD, IgE, IgG, IgM) from which the region was derived. Aportion of the constant region domains makes up the Fc region (the“fragment crystallizable” region), which contains domains responsiblefor the effector functions of an immunoglobulin, such as ADCC(antibody-dependent cell-mediated cytotoxicity), CDC(complement-dependent cytotoxicity) and complement fixation, binding toFc receptors, greater half-life in vivo relative to a polypeptidelacking an Fc region, protein A binding, and perhaps even placentaltransfer (see Capon et al., Nature, 337:525 (1989)). Further, apolypeptide containing an Fc region allows for dimerization ormultimerization of the polypeptide.

A “hinge region” is an amino acid sequence interposed between andconnecting a CD37-specific binding domain and another region (e.g., aCH2 region) in a fusion protein so that the fusion protein is stillcapable of specific binding to CD37 (i.e., with a Ka of at least about10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or10¹³ M⁻¹). In certain embodiments, a hinge region is an immunoglobulinhinge region.

An “immunoglobulin hinge region” refers to a wild type immunoglobulinhinge region or an altered wild type immunoglobulin hinge region.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. The upperhinge region includes amino acids from the carboxyl end of CH1 to thefirst residue in the hinge that restricts motion, generally the firstcysteine residue that forms an interchain disulfide bond between the twoheavy chains. The length of the upper hinge region correlates with thesegmental flexibility of the antibody. The core hinge region containsthe inter-heavy chain disulfide bridges, and the lower hinge regionjoins the amino terminal end of the CH2 domain and includes residues inCH2. Id. The core hinge region of human IgG1 contains the sequenceCys-Pro-Pro-Cys (SEQ ID NO:264) which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility.

A “wild type immunoglobulin hinge region,” as used herein refers to anaturally occurring amino acid sequence interposed between andconnecting CH1 and CH2 regions of a single chain of an antibody. Itcontains the upper hinge region, the core hinge region, and the portionof the lower hinge region that is not part of CH2 region. An exemplarywild type immunoglobulin hinge region is human IgG1 hinge region as setforth in SEQ ID NO:90, in which from its amino terminus to its carboxylterminus, the first ten amino acids (EPKSCDKTHT, SEQ ID NO:263) form theupper hinge region, the next four amino acids (CPPC, SEQ ID NO:264) formthe core hinge region, and the last amino acid (i.e., proline) is thefirst amino acid in the lower hinge region and is not part of CH2.

An “altered wild type immunoglobulin hinge region” or “alteredimmunoglobulin hinge region” refers to (a) a wild type immunoglobulinhinge region with up to 30% amino acid changes (e.g., up to 25%, 20%,15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion ofa wild type immunoglobulin hinge region that is at least 10 amino acids(e.g., at least 12, 13, 14 or 15 amino acids) in length with up to 30%amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acidsubstitutions or deletions), or (c) a portion of a wild typeimmunoglobulin hinge region that comprises the core hinge region (whichmay be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). When analtered wild type immunoglobulin hinge region is interposed between andconnecting a CD37-specific binding domain and another region (e.g., aCH2 region) in a fusion protein, it allows the fusion protein tospecifically bind to CD37 (i.e., with a Ka of at least about 10⁶ M⁻¹,10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹).In certain embodiments, one or more cysteine residues in a wild typeimmunoglobulin hinge region may be substituted by one or more otheramino acid residues (e.g., one or more serine residues). An alteredimmunoglobulin hinge region may alternatively or additionally have aproline residue of a wild type immunoglobulin hinge region substitutedby another amino acid residue (e.g., a serine residue).

A “linker” refers to an amino acid sequence that connects a heavy chainvariable region and a light chain variable region together and providesa spacer function compatible with interaction of the two sub-bindingdomains so that the resulting polypeptide is capable of CD37-specificbinding.

“Derivative” as used herein refers to a chemically or biologicallymodified version of a compound that is structurally similar to a parentcompound and (actually or theoretically) derivable from that parentcompound. Generally, a “derivative” differs from an “analogue” in that aparent compound may be the starting material to generate a “derivative,”whereas the parent compound may not necessarily be used as the startingmaterial to generate an “analogue.” A derivative may have differentchemical or physical properties from the parent compound. For example, aderivative may be more hydrophilic or it may be a mutated sequencehaving altered reactivity (e.g., a CDR having an amino acid change thatalters its affinity for a target) as compared to the parent compound orsequence.

“B-cell associated disorder or disease” refers to aberrant B-cellactivity or activity that deviates from the normal, proper, or expectedcourse. For example, a B-cell associated disorder or disease may includeinappropriate proliferation of cells that have damaged or defective DNAor other cellular components. Aberrant B-cell activity may include cellproliferation characterized by inappropriately high levels of celldivision, inappropriately low levels of apoptosis, or both. Suchdiseases may have, for example, single or multiple local abnormalproliferations of cells, groups of cells or tissue(s), whether cancerousor non-cancerous, benign or malignant. A B-cell associated disorder ordisease may also include aberrant antibody production, such asproduction of autoantibodies, or overproduction of antibodies moredesirable when produced at normal levels. It is also contemplated hereinthat aberrant B-cell activity may occur in certain subpopulations ofB-cells and not in other subpopulations, or may include inappropriatestimulation of T-cells, such as by inappropriate antigen presentation toT-cells or by other B-cells pathway.

“Treatment” or “treating” refers to either a therapeutic treatment orprophylactic/preventative treatment. A therapeutic treatment may improveat least one symptom of disease in an individual receiving treatment ormay delay worsening of a progressive disease in an individual, orprevent onset of additional associated diseases.

A “therapeutically effective amount (or dose)” or “effective amount (ordose)” of a specific binding molecule or compound refers to that amountof the compound sufficient to result in amelioration of one or moresymptoms of the disease being treated. When applied to an individualactive ingredient, administered alone, a therapeutically effective doserefers to that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredserially or simultaneously. The invention specifically contemplates thatone or more specific binding molecules may be administered according tomethods of the invention, each in an effective dose.

“An individual having, or suspected of having, a disease associated withaberrant B-cell activity” is an individual in whom a disease or asymptom of a disorder may be caused by aberrant B-cell activity orB-cell proliferation, may be exacerbated by aberrant B-cell activity, ormay be relieved by regulation of B-cell activity. Examples of suchdiseases are a B-cell malignancy or B-cell cancer (for example, B-celllymphoma, a B-cell leukemia or a B-cell myeloma), a diseasecharacterized by autoantibody production or a disease characterized byinappropriate T-cell stimulation caused by inappropriate B-cell antigenpresentation to T-cells or caused by other pathways involving B-cells.

Additional definitions are provided in the following detaileddescription of the present disclosure.

Humanized CD37-Specific Binding Molecules

In one aspect, the present disclosure provides humanized CD37-specificbinding molecules. These molecules may be in any form that contains ahumanized CD37-specific binding domain, including a humanized anti-CD37antibody, an Fab fragment of a humanized anti-CD37 antibody, a humanizedCD37-specific single chain Fv (scFv), a humanized CD37-specific SMIPprotein, a humanized CD37-specific PIMS protein (a fusion proteincomprising the components of SMIP in the reverse orientation), ahumanized CD37-specific SCORPION protein, and other bi- ormulti-specific binding proteins that comprise at least one humanizedCD37-specific binding domain. Detailed description of SMIP proteins andmethods for making the same may be found, for example, in U.S. PatentPublication Nos. 2003/0133939, 2003/0118592, and 2005/0136049 and WO2005017148. Constructs and methods for making PIMS proteins aredescribed in U.S. application Ser. No. 12/168,875. Methods for makingSCORPION proteins may be found, for example, in PCT ApplicationPublication No. WO 2007/146968. Other exemplary multi-functional fusionproteins may be found, for example, in U.S. Patent ApplicationPublication No. 2006/0051844 and U.S. Pat. No. 7,166,707. Certain bi- ormulti-specific binding proteins may comprise a CD37-specific scFv andone or more other binding domains that are not derived from animmunoglobulin.

Humanized CD37-Specific Binding Domains

An exemplary “humanized CD37-specific binding domain” is animmunoglobulin variable region specific for CD37 that comprises at leastone human framework region.

A “human framework region” refers to a wild type (i.e., naturallyoccurring) framework region of a human immunoglobulin variable region,an altered framework region of a human immunoglobulin variable regionwith less than about 50% (e.g., preferably less than about 45%, 40%,30%, 25%, 20%, 15%, 10%, 5%, or 1%) of the amino acids in the region aredeleted or substituted (e.g., with one or more amino acid residues of anonhuman immunoglobulin framework region at corresponding positions), oran altered framework region of a nonhuman immunoglobulin variable regionwith less than about 50% (e.g., less than 45%, 40%, 30%, 25%, 20%, 15%,10%, or 5%) of the amino acids in the region deleted or substituted(e.g., at positions of exposed residues and/or with one or more aminoacid residues of a human immunoglobulin framework region atcorresponding positions) so that, in one aspect, immunogenicity isreduced.

In certain embodiments, a human framework region is a wild typeframework region of a human immunoglobulin variable region. In certainother embodiments, a human framework region is an altered frameworkregion of a human immunoglobulin variable region with amino aciddeletions or substitutions at one, two, three, four or five positions.In yet certain other embodiments, a human framework region is an alteredframework region of a non-human immunoglobulin variable region withamino acid deletions or substitutions at one, two, three, four or fivepositions.

In certain embodiments, a humanized CD37-specific binding domaincomprises at least one, two, three, four, five, six, seven or eighthuman framework regions (FR) selected from human light chain FR1, humanheavy chain FR1, human light chain FR2, human heavy chain FR2, humanlight chain FR3, human heavy chain FR3, human light chain FR4, and humanheavy chain FR4.

Exemplary human FRs are set forth in SEQ ID NOS:140-146 (human heavychain FR1), SEQ ID NOS:147, 150 and 151 (human heavy chain FR2), SEQ IDNO:154-160 (human heavy chain FR3), SEQ ID NOS: 161-163, 168 and 169(human heavy chain FR4), SEQ ID NOS:170-172, 175, and 177-181 (humanlight chain FR1), SEQ ID NOS:182, 184-188 and 191 (human light chainFR2), SEQ ID NOS:194-198, 203 and 205 (human light chain FR3), and SEQID NOS:206-210 (human light chain FR4). Additional exemplary human FRregions may be found in FR regions of the CD37-specific SMIP proteinsprovided herein, such as CAS-001, CAS-002, CAS-003, or CAS-024.

Human FRs that may be present in CD37-specific binding domains alsoinclude variants of the exemplary FRs provided herein in which one ortwo amino acids of the exemplary FRs have been substituted or deleted.

In certain embodiments, a humanized CD37-specific binding domaincomprises (a) a humanized light chain variable region that comprises ahuman light chain FR1, a human light chain FR2, a human light chain FR3,and a human light chain FR4, and (b) a humanized heavy chain variableregion that comprises a human heavy chain FR1, a human heavy chain FR2,a human heavy chain FR3, and a human heavy chain FR4.

CD37-specific binding domains provided herein also comprise one, two,three, four, five, or six CDRs. Such CDRs may be nonhuman CDRs oraltered nonhuman CDRs selected from CDR1, CDR2 and CDR3 of the lightchain and CDR1, CDR2 and CDR3 of the heavy chain. In certainembodiments, a CD37-specific binding domain comprises (a) a light chainvariable region that comprises a light chain CDR1, a light chain CDR2,and a light chain CDR3, and (b) a heavy chain variable region thatcomprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chainCDR3.

Exemplary CDRs include CDR1 of the light chain as set forth in SEQ IDNO:61 (RASENVYSYLA) or SEQ ID NO:62 (RTSENVYSYLA), CDR1 of the heavychain as set forth in SEQ ID NO:63 (GYMNM), CDR2 of the light chain asset forth in SEQ ID NO:64 (FAKTLAE), CDR2 of the heavy chain as setforth in SEQ ID NO:65 (NIDPYYGGTTYNRKFKG), CDR3 of the light chain asset forth in SEQ ID NO:66 (QHHSDNPWT), CDR3 of the heavy chain as setforth in SEQ ID NO:67 (SVGPFDY), CDR3 of the heavy chain as set forth inSEQ ID NO:68 (SVGPFDS), and CDR3 of the heavy chain as set forth in SEQID NO:69 (SVGPMDY). Preferred light chain CDR1 is SEQ ID NO:61(RASENVYSYLA) and preferred heavy chain CDR3 include SEQ ID NO:68(SVGPFDS) or SEQ ID NO:69 (SVGPMDY).

Additional exemplary CDRs include CDR1 of the light chain as set forthin SEQ ID NO:128 (RTSQNVYSYLA), 129 (RTSESVYSYLA), 130 (RASQSVYSYLA),131 (RASQSVSSYLA) and 132 (RASQSVSYYLA), CDR1 of the heavy chain as setforth in SEQ ID NOS:133 (SYMNM) and 134 (SYWIG), CDR2 of the light chainas set forth in SEQ ID NOS:135 (AASSLQS), 136 (GASTRAT) and 137(DASNRAT), CDR2 of the heavy chain as set forth in SEQ ID NOS:138(IIYPGDSDTRYSPSFQG) and 139 (RIDPSDSYTNYSPSFQG), CDR3 of the light chainas set forth in SEQ ID NO:220 (QHHSDNPWT), and CDR3 of the heavy chainas set forth in SEQ ID NOS:211 (SVGPMDY), 212 (SVGPFDY), 213 (SVGPMDV),214 (SVGPFDS), 215 (SVGPFDP), 216 (SVGPFQH), 217 (SVGPFDV), 218(SVGPFDI) and 219 (SVGPFDL). Further exemplary CDRs include the CDRs inthe CD37-specific SMIP proteins provided herein.

In certain embodiments, CD37-specific binding domains comprise ahumanized light chain variable region that comprises from its aminoterminus to carboxyl terminus: human light chain FR1, light chain CDR1,human light chain FR2, light chain CDR2, human light chain FR3, lightchain CDR3, and human light chain FR4.

In certain embodiments, CD37-specific binding domains comprise ahumanized light chain variable region that comprises from its aminoterminus to carboxyl terminus: human light chain FR1, light chain CDR1as set forth in SEQ ID NO:61 or 62, human light chain FR2, light chainCDR2 as set forth in SEQ ID NO:64, human light chain FR3, light chainCDR3 as set forth in SEQ ID NO:66, and human light chain FR4. In furtherembodiments, CD37-specific binding domains comprise, consist essentiallyof, or consist of a humanized light chain variable region that comprisesfrom its amino terminus to carboxyl terminus: human light chain FR1 asset forth in SEQ ID NO:171, light chain CDR1 as set forth in SEQ IDNO:61, human light chain FR2 as set forth in SEQ ID NO:182, light chainCDR2 as set forth in SEQ ID NO:64, human light chain FR3 as set forth inSEQ ID NO:195, light chain CDR3 as set forth in SEQ ID NO:66, and humanlight chain FR4 as set forth in SEQ ID NO:206. Additional exemplaryhumanized light chains are set forth in SEQ ID NOS:237-240 and includethe light chains in humanized CD37-specific SMIP proteins providedherein.

In certain embodiments, CD37-specific binding domains comprise ahumanized heavy chain variable region that comprises from its aminoterminus to carboxyl terminus: human heavy chain FR1, heavy chain CDR1,human heavy chain FR2, heavy chain CDR2, human heavy chain FR3, heavychain CDR3, and human heavy chain FR4.

In certain embodiments, CD37-specific binding domains comprise ahumanized heavy chain variable region that comprises from its aminoterminus to carboxyl terminus: human heavy chain FR1, heavy chain CDR1as set forth in SEQ ID NO:63, human heavy chain FR2, heavy chain CDR2 asset forth in SEQ ID NO:65, human heavy chain FR3, heavy chain CDR3 asset forth in SEQ ID NO:67, 68 or 69, and human heavy chain FR4. Infurther embodiments, CD37-specific binding domains comprise consistessentially of, or consist of a humanized heavy chain variable regionthat comprises from its amino terminus to carboxyl terminus: human heavychain FR1 as set forth in SEQ ID NO:144, heavy chain CDR1 as set forthin SEQ ID NO:63, human heavy chain FR2 as set forth in SEQ ID NO:151,heavy chain CDR2 as set forth in SEQ ID NO:65, human heavy chain FR3 asset forth in SEQ ID NO:158, heavy chain CDR3 as set forth in SEQ IDNO:67, 68 or 69, and human heavy chain FR4 as set forth in SEQ IDNO:161. Additional exemplary humanized light chains are set forth in SEQID NOS:242-245 and include the light chains in humanized CD37-specificSMIP proteins provided herein.

In certain embodiments, CD37-specific binding domains may be in the formof a Fab or scFv fragment. In a preferred embodiment, the CD37-specificbinding domain is a humanized CD37-specific scFv that comprises a lightchain variable region and a heavy chain variable region joined togethervia a linker. In further embodiments, both the light and heavy chainvariable regions are humanized, and may comprise both a humanized lightchain variable region as set forth in SEQ ID NO:238 and a humanizedheavy chain variable region as set forth in SEQ ID NO:245.

In still further embodiments, only the light or heavy chain variableregion is humanized. For example, CD37-specific binding domains maycomprise a humanized light chain variable region (i.e., a light chainvariable region that comprises at least one human FR) and a nonhumanheavy chain variable chain region (e.g., mouse or rat). Alternatively,CD37-specific binding domains may comprise a nonhuman light chainvariable region (e.g., mouse or rat) and a humanized heavy chainvariable chain region (i.e., a heavy chain variable region thatcomprises at least one human FR). Both types of CD37-specific bindingdomains may be referred to as a “hybrid human-nonhuman CD37-specificbinding domain” or as a “chimeric CD37-specific binding domains.”

In certain embodiments, the carboxyl terminus of the light chainvariable region in a humanized CD37-specific scFv is linked to the aminoterminus of the heavy chain variable region via a linker. Thus, theresulting scFv has from its amino terminus to its carboxyl terminus: thelight chain variable region, the linker, and the heavy chain variableregion. In certain other embodiments, the carboxyl terminus of the heavychain variable region in a humanized CD37-specific scFv is linked to theamino terminus of the light chain variable region via a linker. Thus,the resulting scFv has from its amino terminus to its carboxyl terminus:the heavy chain variable region, the linker, and the heavy chainvariable region.

In certain embodiments, the linkers have 5-30 amino acids, such as 15-25amino acids. In certain embodiments, the linkers comprises(Gly_(n)Ser)_(m), wherein n and m may be an integer independentlyselected from 1 to 5. For example, in certain embodiments, n is 4, and mis 1, 2, 3, 4 or 5. In certain embodiments, one or two amino acids otherthan Gly or Ser may be present at the amino terminus, carboxyl terminusor both termini. In certain other embodiments, one or two amino acidsother than Gly or Ser may be used to substitute a Gly or Ser in a linkerthat comprises (Gly_(n)Ser)_(m) with m and n as defined above. Anexemplary linker has the sequence (Gly₄S)₅ as set forth in SEQ IDNO:229. Additional exemplary linker sequences are set forth in SEQ IDNOS:225-228.

In certain embodiments, humanized CD37-specific binding domains orCD37-specific binding molecules competes with G28-1 mAb for binding toCD37. In other words, in such embodiments, CD37 binding of G28-1 mAb isreduced in the presence of other CD37-specific binding domains (such asanti-CD37 monoclonal antibodies) or CD37-specific binding moleculescompared to CD37 binding of G28-1 mAb in the absence of CD37-specificbinding domains or

CD37-specific binding molecules. Competitive binding assays are known inthe art, such as those described in the Examples 4-6, and may be used todetermine whether a given CD37-specific binding domain or CD37-specificbinding molecule is capable of competing with G28-1 mAb for binding toCD37.

Humanized CD37-Specific SMIP Polypeptides

In certain embodiments, CD37-specific binding molecules areCD37-specific small modular immunopharmaceutical (SMIP) polypeptides.SMIP proteins are binding domain-immunoglobulin fusion proteins thattypically comprise from their amino termini to carboxyl termini: abinding domain derived from an immunoglobulin (e.g., a scFv), a hingeregion, and an effector domain (e.g., IgG CH2 and CH3 regions). Inpreferred embodiments, the CD37-specific binding SMIP polypeptides arehumanized.

The hinge region of a humanized CD37-specific binding SMIP polypeptidemay be an immunoglobulin hinge region. In certain embodiments, the hingeregion is a wild type immunoglobulin hinge region, such as an IgG hinge,IgA hinge, IgD hinge, IgE hinge or a fragment thereof (e.g., 4 to 20 or5 to 15 amino acids in length) that comprises a core hinge region. Incertain preferred embodiments, a hinge region may be an antibody hingeregion selected from human IgG1, human IgG2, human IgG3, human IgG4, orfragments or variants thereof. In some embodiments, the hinge region isa wild type immunoglobulin hinge region or portion thereof, such as ahuman immunoglobulin hinge region. Exemplary hinges for such embodimentsare wild type human IgG1 hinge region as set forth in SEQ ID NO:90, wildtype human IgA1 hinge as set forth in SEQ ID NO:115, wild type humanIgA2 hinge as set forth in SEQ ID NO:116, wild type human IgG3 hinge asset forth in SEQ ID NO:118, a portion of human IgG3 hinge as set forthin SEQ ID NO:258, and human IgD hinge as set forth in SEQ ID NO:127. Incertain embodiments, one or more amino acid residues may be added at theamino- or carboxy-terminus of a wild type immunoglobulin hinge region aspart of fusion protein construct design. Such amino acid residues arereferred to as “junction amino acids” (see Table 4).

In certain embodiments, the hinge region is an altered (mutated) wildtype immunoglobulin hinge region, such as an altered wild type IgGimmunoglobulin hinge region, or an altered portion of a wild typeimmunoglobulin hinge region. For example, the wild type human IgG1 hingeregion contains three cysteine residues—the most N-terminal cysteine isreferred to the first cysteine, whereas the most C-terminal cysteine inthe hinge region is the third cysteine. In certain embodiments, themutated human IgG1 hinge region has only two cysteine residues, such asa human IgG1 hinge region with the first cysteine substituted by aserine. In certain other embodiments, the mutated human IgG1 hingeregion has only one cysteine residue. In certain embodiments, theproline C-terminal to the third cysteine in the human IgG1 hinge regionis substituted, for example, by a serine. Exemplary mutated human IgG1hinge regions are as set forth in SEQ ID NOS:92, 94, 102, 104, 255, 256,106, 108, 257, 96, 110, 112, 98, and 100. Exemplary mutated portions ofhuman IgG3 hinge regions are as set forth in SEQ ID NOS:120, 126,259-261, 122, and 124. In certain embodiments, one or more amino acidresidues may be added at the amino-or carboxy-terminus of a mutatedimmunoglobulin hinge region as part of fusion protein construct design.Examples of such modified hinge regions are indicated in italics in SEQID NOS:231-235.

In certain embodiments, a hinge region comprises or has a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to a wild type immunoglobulin hinge region, such as a wildtype human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD and IgE hinges.

In further embodiments, altered hinge regions can be based on a wildtype immunoglobulin hinge region (e.g., an IgG1 hinge region) andcontain one or more (e.g., 1, 2, 3, or 4) insertions, one or more (e.g.,1, 2, 3, or 4) deletions, one or more (e.g., 1, 2, 3, or 4) amino acidsubstitutions (e.g., conservative amino acid substitutions ornon-conservative amino acid substitutions), or a combination of theabove-noted mutations, when compared with the wild type immunoglobulinhinge region, but provided that the modified hinge retains theflexibility or rigidity suitable for properly orienting the bindingdomain of a fusion binding protein to interact with its target. Theinsertion(s), deletion(s) or substitution(s) may be anywhere in the wildtype immunoglobulin hinge region, including at the amino- orcarboxy-terminus or both.

As described herein, CD37-specific SMIP polypeptides may comprise animmunoglobulin C_(H2) region. In certain embodiments, the immunoglobulinC_(H2) region is a wild type immunoglobulin C_(H2) region, such as awild type human immunoglobulin C_(H2) region, including wild type humanIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM C_(H2) regions. Incertain embodiments, the immunoglobulin C_(H2) region is a human IgG1C_(H2) region.

In certain other embodiments, the immunoglobulin C_(H2) region is analtered wild type immunoglobulin C_(H2) region. For example, the alteredwild type immunoglobulin C_(H2) region may be a human IgG1 C_(H2) regionbut with one, two, three, four or five mutations at positions 234 to238, 253, 279, 310, 318, 320, 322, and 331 (EU numbering, Ward et al.,1995 Therap. Immunol. 2:77-94). The mutations in such positions reduceor eliminate the antibody-dependent cell-mediated cytotoxicity (ADCC)activity, Fc receptor-binding capability, and/or complement fixation.

As described herein, a humanized CD37-specific SMIP polypeptides maycomprise an immunoglobulin C_(H3) region. In certain embodiments, animmunoglobulin C_(H3) region polypeptide is a wild type immunoglobulinC_(H3) region polypeptide, including a wild type C_(H3) region of anyone of the various immunoglobulin isotypes (e.g., IgA, IgD, IgG1, IgG2,IgG3, IgG4, IgE, or IgM) from various species (i.e., human, mouse, rator other mammals). In other embodiments, an immunoglobulin C_(H3) regionpolypeptide is a mutated immunoglobulin C_(H3) region polypeptide. Themutations in the immunoglobulin C_(H3) region may be at one or morepositions that are involved in complement fixation, such as at H433 orN434.

In certain embodiments, a humanized CD37-specific SMIP polypeptides maycontain one or more additional regions. Such additional regions may be aleader sequence at the amino-terminus for secretion of an expressed SMIPpolypeptide, an additional Fc sub-region (e.g., a wild type or mutatedC_(H4) region of IgM or IgE), a tail sequence at its carboxy-terminusfor identification or purification purposes (e.g., epitope tags fordetection or purification, including a 6-Histidine tag or a FLAGepitope), or additional amino acid residues that arise from use ofspecific expression systems. Exemplary leader peptides of thisdisclosure include natural leader sequences or others, such as those asset forth in SEQ ID NOS:223 and 224.

This disclosure includes CD37-specific SMIP polypeptides that exhibit atleast 80 percent identity (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%,95%, 96%, 97%, 98% or 99%) to the polypeptide set forth in SEQ ID NO:2,wherein the CD37-specific SMIP polypeptide binds CD37. In furtherembodiments, such polypeptides having at least 80% identity with SEQ IDNO:2 may be further humanized. Exemplary humanized CD37-specific SMIPpolypeptides comprise, consist essential of, or consist of any aminoacid sequence selected from the group consisting of SEQ ID NOS:6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 52, 80, 82, 84, 86, 88, and 222 in which the leader sequences aredeleted, as well as SEQ ID NOS:247-254 and 266-269.

“Sequence identity,” as used herein, refers to the percentage of aminoacid residues in one sequence that are identical with the amino acidresidues in another reference polypeptide sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. The percentage sequenceidentity values are generated by the NCBI BLAST2.0 software as definedby Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: a new generationof protein database search programs”, Nucleic Acids Res. 25:3389-3402,with the parameters set to default values. In a preferred embodiment,the present disclosure provides a humanized CD37-specific SMIPpolypeptide that comprises from amino terminus to carboxyl terminus: ahumanized heavy chain variable region (V_(H)), a (G₄S)₅ linker (SEQ IDNO:229), a humanized light chain variable region (V_(L)), an alteredIgG1 hinge, a human IgG1 CH2 region, and a human IgG1 CH3 region. Thehumanized heavy chain variable region comprises from its amino terminusto its carboxyl terminus: a human heavy chain FR1, a heavy chain CDR1 asset forth in SEQ ID NO:63, a human heavy chain FR2, a CDR2 as set forthin SEQ ID NO:65, a human heavy chain FR3, CDR3 as set forth in SEQ IDNO:67, 68 or 69, and a human heavy chain FR4. The humanized light chainvariable region comprises from its amino terminus to its carboxylterminus: a human light chain FR1, a light chain CDR1 as set forth inSEQ ID NO:61 or 62, a human light chain FR2, a light chain CDR2 as setforth in SEQ ID NO:64, a human light chain FR3, and a light chain CDR3as set forth in SEQ ID NO:66, and a human light chain FR4.

In some of the above preferred embodiments, the human heavy chain FR1,FR2, and FR3 comprise SEQ ID NOS:144, 151, and 158, respectively, andthe heavy chain FR4 comprises SEQ ID NO:161 or 162. In further preferredembodiments, the human light chain FR1, FR2, FR3, and FR4 comprise SEQID NOS:171, 182, 195, and 206, respectively. Alternatively, both theheavy and light chains contain these sequences.

The CAS-024 SMIP protein is unexpectedly (1) expressed at up to about25-fold higher levels than other humanized versions of CAS-006 (such asCAS-002, CAS-003; see Examples 2 and 5), (2) capable of binding CD37 aswell as CAS-006 while other humanized versions do not (see Examples 4and 5), and (3) produced as a homogenous population of molecules ascompared the heterogenous nature of other humanized versions (seeExample 3) In a preferred embodiment, the instant disclosure provides aCD37-specific binding protein that comprises or consists of CAS-024 (SEQID NO:253). In particular, this humanized CD37-specific binding moleculehas substantially the same CD37 binding affinity as its parent chimericmolecule (CAS-006, SMIP protein having the immunoglobulin variableregions from mouse anti-human CD37 monoclonal antibody G28-1) incontrast to other humanized molecules, is expressed at high levelscompared to other humanized molecules, and/or shows a high degree ofhomogeneity when purified, for example, via size exclusionchromatography (SEC) in contrast to other humanized molecules. Inaddition, this CAS-024 CD37-specific binding molecule has been shown tobe effective in inhibiting tumor growth and causing long term tumorregression.

The disclosure also includes an isolated nucleic acid moleculecomprising a nucleotide sequence encoding humanized CD37-specificbinding molecules and the components thereof, including human orhumanized FRs, CDRs, humanized light chain variable regions, humanizedheavy chain variable regions, humanized scFv, and humanized SMIPpolypeptides. Exemplary isolated nucleic acid molecules that encodehumanized CD37-specific SMIP polypeptides include those that compriseSEQ ID NOS:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 51, 79, 81, 83, 85, 87, and 221. In oneembodiment, the disclosure includes vectors that comprise these nucleicacid molecules and host cells that comprise the vectors.

The disclosure also includes processes of producing the polypeptidesdescribed herein, comprising culturing the host cells under suitableconditions to express the polypeptides, and optionally isolating thepolypeptides from the culture.

Compounds Useful for Combination Therapy

The present disclosure also provides a combination therapy using any ofthe CD37-specific binding molecules known in the art or disclosed hereinand bifunctional chemotherapeutics (e.g., bendamustine).

The CD37-specific binding molecules useful for the combination therapymay be in any forms that contain a CD37-specific binding domain,including an anti-CD37 antibody, an Fab fragment of anti-CD37 antibody,a CD37-specific single chain Fv (scFv), a CD37-specific SMIP, aCD37-specific PIMS, a CD37-specific SCORPION, and other bi- ormulti-specific binding proteins that comprise at least one CD37-specificbinding protein.

In certain embodiments, the CD37-specific binding molecules useful forcombination therapy with a bifunctional chemotherapeutic areCD37-specific antibodies. Such antibodies include those used forcharacterizing the CD37 antigen in the Thrid HLDA Workshop, i.e., HD28,G28-1, HH1, B114, WR17 and F93G6 (see, Ling and MacLennan, pp. 302-335in Leucocyte Typing III. White Cell Differentiation Antigens, OxfordUniversity Press (1987)). Other CD37-specific antibodies useful for thecombination therapy include RFB-7, Y29/55, MB-1, M-B371, M-B372 andIPO-24 (see, Moldenhaurer, J. Biol., Regul. Homeost. Agents, 14: 281-283(2000), stating that all these antibodies recognize only one CD37epitope, and Schwartz-Albiez et al., 14: 905-914 (1988), indicating thatthe epitope is situated in the carbohydrate moiety of CD37). AnotherCD37-specific antibody that may be used in combination therapy is S-B3(Biosys).

In certain embodiments, the CD37-specific binding molecules useful forcombination therapy with a bifunctional chemotherapeutic areCD37-specific SMIP polypeptides. An exemplary SMIP polypeptide comprisesSEQ ID NO:2. Additional exemplary SMIP polypeptides include thosedescribed in WO 2005017148, such as (1) G28-1 scFv (SSS-S) H WCH2 WCH3comprising a G28-1 scFv, an altered human IgG1 hinge in which all threecysteine residues and a proline carboxyl terminus to the third cysteinein a human IgG1 hinge region are mutated to serine residues, and wildtype human IgG1 CH2 and CH3 domains; (2) G28-1 scFv IgAH WCH2 WCH3comprising a G28-1 scFv, a portion of human IgA hinge, and human IgG1CH2 and CH3 domains; (3) G28-1 scFv VHL11S (SSS-S) H WCH2 CH3 comprisinga G28-1 scFv, an altered human IgG1 hinge in which all three cysteineresidues and a proline carboxyl terminus to the third cysteine in thehinge region are mutated to serine residues, and human IgG1 CH2 and CH3domains, wherein the leucine at position 11 of the heavy chain variableregion is substituted with a serine; (4) G28-1 scFv VH L115 (CSS-S) HWCH2 CH3 comprising a G28-1 scFv, an altered human IgG1 hinge in whichthe cysteine residues at the second and third positions and a prolinecarboxyl terminus to the third cysteine are substituted with serineresidues, and human IgG1 CH2 and CH3 domains, wherein the leucine atposition 11 of the heavy chain variable region is substituted with aserine; (5) G28-1 scFv VHL11S (CSC-S) H WCH2 CH3 comprising a G28-1scFv, an altered human IgG1 hinge in which the cysteine residue at thesecond position and a proline carboxyl terminus to the cysteine at thethird position were substituted with serine residues, and human IgG1 CH2and CH3 domains, wherein the leucine at position 11 of the heavy chainvariable region is substituted with a serine; (6) G28-1 scFv VH11S(SSC-P) H WCH2 WCH3 comprising a G28-1 scFv, an altered human IgG1 hingein which the first and second cysteine residues in the hinge region aremutated to serine residues, and human IgG1 CH2 and CH3 domains, whereinthe leucine at position 11 of the heavy chain variable region issubstituted with a serine; (7) G28-1 scFv VH115 (SCS-S) H WCH2 WCH3comprising a G28-1 scFv, an altered human IgG1 hinge in which the firstand third cysteine residues and a proline carboxyl terminus to the thirdcysteine in the hinge regions are mutated to serine residues, and humanIgG1 CH2 and CH3 domains, wherein the leucine at position 11 of theheavy chain variable region is substituted with a serine; (8) G28-1 scFvVHL11S (CCS-P) H WCH2 WCH3 comprising a G28-1 scFv, an altered humanIgG1 hinge in which the third cysteine residue in the hinge region issubstituted with a serine, and human IgG1 CH2 and CH3 domains, whereinthe leucine at position 11 of the heavy chain variable region issubstituted with a serine (9) G28-1scFv VHL11S (SCC-P) H WCH2 WCH3comprising a G28-1 scFv, an altered human IgG1 hinge in which the firstcysteine is substituted with a serine, and human CH2 and CH3 domains,wherein the leucine at position 11 of the heavy chain variable region issubstituted with a serine; (10) G28-1 scFv VH L11S mIgE CH2 CH3 CH4,comprising a G28-1 scFv and mouse IgE CH2, CH3 and CH4 regions, whereinthe leucine at position 11 of the heavy chain variable region issubstituted with a serine; (11) G28-1 scFv VH L11S mIgA WIgACH2 T4CH3,comprising a G28-1 scFv, a mouse IgA hinge, and a wild type IgA CH2 anda truncated IgA CH3 domain lacking the 4 carboxy amino acids GTCY (SEQID NO:265); (12) G28-1 scFv VHL11S hIgE CH2 CH3 CH4, comprising a G28-1scFv and human IgE CH2, CH3 and CH4 regions, wherein the leucine atposition 11 of the heavy chain variable region is substituted with aserine; and (13) G28-1 scFv VHL11S hIgAH WIgACH2 TCH3 comprising a G28-1scFv, a portion of human IgA hinge, a wild type IgA CH2 and a truncatedIgA CH3 domain lacking the 4 carboxy amino acids GTCY (SEQ ID NO:265),wherein the leucine at position 11 of the heavy chain variable region issubstituted with a serine.

In certain embodiments, CD37-specific binding molecules useful forcombination therapy with a bifunctional chemotherapeutic are humanizedCD37-specific binding molecules described herein, including humanizedanti-CD37 antibodies, Fab fragments of humanized anti-CD37 antibody,humanized CD37-specific PIMS protein, humanized CD37-specific SCORPIONprotein, and other bi- or multi-specific binding proteins that compriseat least one humanized CD37-specific binding protein, especiallyhumanized CD37-specific single chain Fv (scFv) and humanizedCD37-specific SMIP polypeptides.

Certain CD37-specific binding molecules contemplated in this disclosurehave affinities for CD37 of about 0.5 to about 10 nM. Anothercharacteristic of certain CD37-binding molecules contemplated in thisdisclosure is that they exhibit a half life in circulation of about 5 toabout 30 days.

In certain embodiments, CD37-specific binding molecules are capable ofcompeting with G28-1 mAb in CD37-specific binding.

Bendamustine(4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid)is a nitrogen mustard with alkylator and antimetabolite activities.Bendamustine has both an alkylating group and a benzimidazole ring. Thealkylating group allows bendamustine metabolites to alkylate andcrosslink macromolecules, resulting in DNA, RNA and protein synthesisinhibition, and subsequently apoptosis. The benzimidazole ring may allowbendamustine to act as a purine analogue. Bendamustine hydrochloride hastrade names TREANDA® and RIBOMUSTIN®.

Although bendamustine or its salts are preferred therapeutic agents thatmay be used in combination with CD37-specific binding molecules, othertherapeutic agents that both comprise one or more alkylating groups andare capable of functioning as a purine analogue may also be used incombination with CD37-specific binding molecules according to thepresent disclosure.

The term “alkylating group,” as used herein, refers to a group thatenables the compound comprising this group to attach an alkyl group toDNA. The compound that comprises an alkylating group may be referred toas an “alkylating agent.” In certain embodiments, alkylating agents arenitrogen mustards.

The term “purine analogue” refers to an antimetabolite that mimics thestructure of metabolic purines (e.g., adenine and guanine) and has one,two, three or four substituents at the purine ring that differ frommetabolic purines. Exemplary purine analogues include azathioprine,mercaptopurine, thioguanine, fludarabine, pentostatin and cladribine.

A therapeutic agent is “capable of functioning as a purine analogue” ifit possesses at least one function of a purine analogue. Exemplaryfunctions of purine analogues include interference with or inhibition ofpurine nucleotide synthesis, purine nucleotide metabolism, nucleic acidsynthesis, nucleic acid processing, or nucleic acid function, such asinhibiting ribonucleotide reductase, DNA polymerase, adenosinedeaminase, and being incorporated into DNA or RNA.

Compositions and Methods

In one aspect, the present disclosure provides a method for reducing Bcells or treating a disease associated with aberrant B cell activitycomprising administering to a subject in need thereof (i.e., anindividual having or suspected of having a disease associated withaberrant B-cell activity) an effective amount of a humanizedCD37-specific binding molecule provided herein (e.g., CAS-024).

In another aspect, the present disclosure provides a method for reducingB cells or treating a disease associated with aberrant B cell activitycomprising administering to a subject in need thereof an effectiveamount of a CD37-specific binding molecule (e.g., CAS-024) and abifunctional chemotherapeutic (e.g., bendamustine). As described above,CD37-specific binding molecules useful for combination therapy with abifunctional chemotherapeutic are not limited to humanized CD37-specificbinding molecules, but include other CD37-specific binding moleculesthat have not been humanized.

In one embodiment, a composition comprising a CD37 therapeutic and abifunctional chemotherapeutic act synergistically in reducing B cells ortreating a disease associated with aberrant B cell activity. Two or morecompounds that act synergistically interact such that the combinedeffect of the compounds is greater than the sum of the individualeffects of each compound when administered alone (see, e.g., Berenbaum,Pharmacol. Rev. 41:93, 1989). For example, an interaction between smallmodular immunopharmaceutical that targets CD37 and another agent orcompound may be analyzed by a variety of mechanistic and empiricalmodels (see, e.g., Ouzounov et al., Antivir. Res. 55:425, 2002). Acommonly used approach for analyzing the interaction between acombination of agents employs the construction of isoboles (iso-effectcurves, also referred to as isobolograms), in which the combination ofagents (d_(a), d_(b)) is represented by a point on a graph, the axes ofwhich are the dose-axes of the individual agents (see, e.g., Ouzounov etal., supra; see also Tallarida, J. Pharmacol. Exp. Therap. 298:865,2001).

Another method for analyzing drug-drug interactions (antagonism,additivity, synergism) known in the art includes determination ofcombination indices (CI) according to the median effect principle toprovide estimates of 10₅₀ values of compounds administered alone and incombination (see, e.g., Chou. In Synergism and Antagonism Chemotherapy.Eds. Chou and Rideout. Academic Press, San Diego Calif., pages 61-102,1991; CalcuSyn™ software). A CI value of less than one representssynergistic activity, equal to one represents additive activity, andgreater than one represents antagonism.

Still another exemplary method is the independent effect method(Pritchard and Shipman, Antiviral Res. 14:181, 1990; Pritchard andShipman, Antiviral Therapy 1:9, 1996; MACSYNERGY™ II software,University of Michigan, Ann Arbor, Mich.). MACSYNERGY™ II softwareallows a three-dimensional (3-D) examination of compound interactions bycomparing a calculated additive surface to observed data to generatedifferential plots that reveal regions (in the form of a volume) ofstatistically greater than expected (synergy) or less than expected(antagonism) compound interactions. For example, a compositioncomprising a CD37-specific binding molecule and a bifunctionalchemotherapeutic alters viral replication will be considered to havesynergistic activity or have a synergistic effect when the volume ofsynergy produced as calculated by the volume of the synergy peaks ispreferably about 15% greater than the additive effect (that is, theeffect of each agent alone added together), or preferably about a 2-foldto 10-fold greater than the additive effect, or preferably about a3-fold to 5-fold or more greater than the additive effect.

In further embodiments, a CD37-specific binding molecule and abifunctional chemotherapeutic can be administered to act synergisticallyin the treatment of B-cell malignancies or B-cell cancers. B-cellmalignancies or B-cell cancers include B-cell lymphomas [such as variousforms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) or centralnervous system lymphomas], leukemias [such as acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemiaand chronic myoblastic leukemia] and myelomas (such as multiplemyeloma). Additional B cell cancers include small lymphocytic lymphoma,B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenicmarginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma ofbone, extraosseous plasmacytoma, extra-nodal marginal zone B-celllymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginalzone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuselarge B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma, Burkittlymphoma/leukemia, B-cell proliferations of uncertain malignantpotential, lymphomatoid granulomatosis, and post-transplantlymphoproliferative disorder.

Burkitt's lymphoma (or “Burkitt's B cell malignancy”, or “Burkitt'stumor”, or “Malignant lymphoma, Burkitt's type”) is a cancer of thelymphatic system (in particular, B lymphocytes). It can be divided intothree main clinical variants: the endemic, the sporadic and theimmunodeficiency-associated variants.

Non-Burkitt's B cell malignancies include, but are not limited to,B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma,B-cell prolymphocytic leukemia, an acute lymphoblastic leukemia (ALL),lymphoplasmacytic lymphoma (including, but not limited to, Waldenstrom'smacroglobulinemia), marginal zone lymphomas (including, but not limitedto, splenic marginal zone B-cell lymphoma, nodal marginal zone lymphoma,and extranodal marginal zone B-cell lymphoma of mucosa-associatedlymphoid tissue (MALT) type), hairy cell leukemia, plasma cellmyeloma/plasmacytoma, follicular lymphoma, mantle cell lymphoma (MCL),diffuse large cell B-cell lymphoma, transforming large B cell lymphoma,mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma,primary effusion lymphoma, and non-Burkitt's non-Hodgkins lymphoma(NHL).

Disorders characterized by autoantibody production are often consideredautoimmune diseases. Autoimmune diseases include, but are not limitedto: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, polychondritis, psoriatic arthritis, psoriasis,dermatitis, polymyositis/dermatomyositis, inclusion body myositis,inflammatory myositis, toxic epidermal necrolysis, systemic sclerodermaand sclerosis, CREST syndrome, responses associated with inflammatorybowel disease, Crohn's disease, ulcerative colitis, respiratory distresssyndrome, adult respiratory distress syndrome (ARDS), meningitis,encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions,eczema, asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis,lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergicencephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham'schorea, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including Wegener's granulomatosis andChurg-Strauss disease, agranulocytosis, vasculitis (includinghypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis),aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia, purered cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, central nervous system (CNS) inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection,graft versus host disease (GVHD), pemphigoid bullous, pemphigus,autoimmune polyendocrinopathies, seronegative spondyloarthropathies,Reiter's disease, stiff-man syndrome, giant cell arteritis, immunecomplex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediatedneuropathy, idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes also referred to as insulin-dependent diabetes mellitus(IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barre' Syndrome, large vesselvasculitis (including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), polyarteritis nodosa (PAN) ankylosingspondylitis, Berger's disease (IgA nephropathy), rapidly progressiveglomerulonephritis, primary biliary cirrhosis, Celiac sprue (glutenenteropathy), cryoglobulinemia, cryoglobulinemia associated withhepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease,familial Mediterranean fever, microscopic polyangiitis, Cogan'ssyndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans,autoimmune thyroid disease (such as Graves' disease and Hashimoto'sthyroiditis), Sjogren's syndrome, and idiopathic inflammatory myopathy(IIM), including dermatomyositis (DM) and polymyositis (PM). The aboveautoimmune diseases may also be treated with humanized CD37-specificbinding molecules or with the combination of CD37-specific bindingmolecules and a bifunctional chemotherapeutic.

In one aspect of the disclosure, a humanized CD37-specific bindingmolecule or a combination of a CD37-specific binding molecule with abifunctional chemotherapeutic is administered in a pharmaceuticalcomposition. To administer a humanized CD37-specific binding molecule ora combination of a CD37-specific binding molecule with a bifunctionalchemotherapeutic to human or test animals, it is preferable to formulatethe binding molecule or the combination in a composition comprising oneor more pharmaceutically acceptable carriers. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce allergic, or other adversereactions when administered using routes well-known in the art, asdescribed below. “Pharmaceutically acceptable carriers” include any andall clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. In addition, compounds may form solvates with wateror common organic solvents. Such solvates are contemplated as well.

Pharmaceutical compositions of the present disclosure containing ahumanized CD37-specific binding molecule or a combination of aCD37-specific binding molecule with a bifunctional chemotherapeutic usedin a method of the disclosure may contain pharmaceutically acceptablecarriers or additives depending on the route of administration. Examplesof such carriers or additives include water, a pharmaceutical acceptableorganic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, acarboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium,sodium alginate, water-soluble dextran, carboxymethyl starch sodium,pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic,casein, gelatin, agar, diglycerin, glycerin, propylene glycol,polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid,human serum albumin (HSA), mannitol, sorbitol, lactose, apharmaceutically acceptable surfactant and the like. Additives used arechosen from, but not limited to, the above or combinations thereof, asappropriate, depending on the dosage form of the present disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the antibody to be administered canbe prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers

A variety of aqueous carriers, e.g., water, buffered water, 0.4% saline,0.3% glycine, or aqueous suspensions may contain the active compound inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

A CD37-specific binding molecule, a combination of a CD37-specificbinding molecule with a bifunctional chemotherapeutic, or a compositioncomprising the binding molecule or the combination can be lyophilizedfor storage and reconstituted in a suitable carrier prior to use. Thistechnique has been shown to be effective with conventionalimmunoglobulins. Any suitable lyophilization and reconstitutiontechniques can be employed. It will be appreciated by those skilled inthe art that lyophilization and reconstitution can lead to varyingdegrees of activity loss and that use levels may have to be adjusted tocompensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of CD37-specific binding molecule or bifunctionalchemotherapeutic in these formulations can vary widely, for example fromless than about 0.5%, usually at or at least about 1% to as much as 15or 20% by weight and will be selected primarily based on fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. Thus, a typical pharmaceutical composition forparenteral injection could be made up to contain 1 mL sterile bufferedwater, and 50 mg of antibody. A typical composition for intravenousinfusion could be made up to contain 250 mL of sterile Ringer'ssolution, and 150 mg of antibody. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed., Mack PublishingCompany, Easton, Pa. (1980). An effective dosage of CD37-specificbinding molecules (including humanized CD37-specific binding molecules)is within the range of 0.01 mg to 1000 mg per kg of body weight peradministration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial or antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, or the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544, 1993).

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the disclosure are well balanced,thereby enhancing their utility for both in vitro and especially in vivouses, while other compositions lacking such balance are of substantiallyless utility. Specifically, compositions contemplated for use in thedisclosure have an appropriate degree of solubility in aqueous mediawhich permits absorption and bioavailability in the body, while alsohaving a degree of solubility in lipids which permits the compounds totraverse the cell membrane to a putative site of action. Thus, antibodycompositions contemplated are maximally effective when they can bedelivered to the site of target antigen activity.

In one aspect, methods of the disclosure include a step ofadministration of a CD37-specific binding molecule composition. Incertain embodiments, the combinations of compounds may be administeredconcurrently, together in the same pharmaceutically acceptable carrier,or separately (but concurrently). In other embodiments, the CD37immunotherapeutic (i.e., the CD37-specific binding molecule) and abifunctional chemotherapeutic can be administered sequentially, in anyorder and in any combination.

The binding molecule, bifunctional chemotherapeutic, or combinationcompositions may be administered orally, topically, transdermally,parenterally, by inhalation spray, vaginally, rectally, or byintracranial injection, or any combination thereof. In one embodiment,both the CD37-specific binding molecule and the bifunctionalchemotherapeutic are administered parenterally, either concurrently orsequentially. The term parenteral, as used herein, includes subcutaneousinjections, intravenous, intramuscular, intracisternal injection, orinfusion techniques. Administration by intravenous, intradermal,intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar,intrapulmonary injection and or surgical implantation at a particularsite is contemplated as well. Generally, compositions are essentiallyfree of pyrogens, as well as other impurities that could be harmful tothe recipient. Injection, especially intravenous, is preferred.

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the disclosure implanted near the cancer.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, weekly or monthly.

Binding molecule, bifunctional chemotherapeutic, or combinationcompositions of this disclosure may comprise one or more than onebinding molecule, bifunctional chemotherapeutic, or any combinationthereof. Also contemplated by the present disclosure is theadministration of binding molecule, bifunctional chemotherapeutic, orcombination compositions in conjunction with a further therapeuticagent. Further therapeutic contemplated by the disclosure are listed inparagraphs below.

A further therapeutic agent may be a B-cell-associated molecule.

Other B-cell-associated molecules contemplated by the disclosure includebinding molecules which bind to B-cell surface molecules that are notCD37. B-cell-associated molecules, include CD19 (B-lymphocyte antigenCD19, also referred to as B-lymphocyte surface antigen B4, or Leu-12),CD20, CD21, CD22 (B-cell receptor CD22, also referred to as Leu-14,B-lymphocyte cell adhesion molecule, or BL-CAM), CD23, CD40 (B-cellsurface antigen CD40, also referred to as Tumor Necrosis Factor receptorsuperfamily member 5, CD40L receptor, or Bp50), CD80 (T lymphocyteactivation antigen CD80, also referred to as Activation B7-1 antigen,B7, B7-1, or BB1), CD86 (T lymphocyte activation antigen CD86, alsoreferred to as Activation B7-2 antigen, B70, FUN-1, or BU63), CD137(also referred to as Tumor Necrosis Factor receptor superfamily member9), CD152 (also referred to as cytotoxic T-lymphocyte protein 4 orCTLA-4), L6 (Tumor-associated antigen L6, also referred to asTransmembrane 4 superfamily member 1, Membrane component surface marker1, or M3S1), CD30 (lymphocyte activation antigen CD30, also referred toas Tumor Necrosis Factor receptor superfamily member 8, CD30L receptor,or Ki-1), CD50 (also referred to as Intercellular adhesion molecule-3(ICAM3), or ICAM-R), CD54 (also referred to as Intercellular adhesionmolecule-1 (ICAM1), or Major group rhinovirus receptor), B7-H1 (ligandfor an immunoinhibitory receptor expressed by activated T cells,B-cells, and myeloid cells, also referred to as PD-L1; see Dong, et al.,“B7-H1, a third member of the B7 family, co-stimulates T-cellproliferation and interleukin-10 secretion,” Nat. Med., 5:1365-1369(1999), CD134 (also referred to as Tumor Necrosis Factor receptorsuperfamily member 4, OX40, OX40L receptor, ACT35 antigen, orTAX-transcriptionally activated glycoprotein 1 receptor), 41 BB (4-1BBligand receptor, T-cell antigen 4-1 BB, or T-cell antigen ILA), CD153(also referred to as Tumor Necrosis Factor ligand superfamily member 8,CD30 ligand, or CD30-L), CD154 (also referred to as Tumor NecrosisFactor ligand superfamily member 5, TNF-related activation protein,TRAP, or T cell antigen Gp39), Toll receptors, or the like.

Examples of chemotherapeutic agents contemplated as further therapeuticagents include alkylating agents, such as nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, andchlorambucil); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU)); ethyleneimines and methyl-melamines (e.g.,triethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), andhexamethylmelamine (HMM, altretamine)); alkyl sulfonates (e.g.,buslfan); and triazines (e.g., dacabazine (DTIC)); antimetabolites, suchas folic acid analogues (e.g., methotrexate, trimetrexate, andpemetrexed (multi-targeted antifolate)); pyrimidine analogues (such as5-fluorouracil (5-FU), fluorodeoxyuridine, gemcitabine, cytosinearabinoside (AraC, cytarabine), 5-azacytidine, and2,2′-difluorodeoxycytidine); and purine analogues (e.g,6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin(pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate,2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I topoisomeraseinhibitors such as camptothecin (CPT), topotecan, and irinotecan;natural products, such as epipodophylotoxins (e.g., etoposide andteniposide); and vinca alkaloids (e.g., vinblastine, vincristine, andvinorelbine); anti-tumor antibiotics such as actinomycin D, doxorubicin,and bleomycin; radiosensitizers such as 5-bromodeozyuridine,5-iododeoxyuridine, and bromodeoxycytidine; platinum coordinationcomplexes such as cisplatin, carboplatin, and oxaliplatin; substitutedureas, such as hydroxyurea; and methylhydrazine derivatives such asN-methylhydrazine (MIH) and procarbazine.

Further therapeutic agents contemplated by this disclosure for treatmentof autoimmune diseases are referred to as immunosuppressive agents,which act to suppress or mask the immune system of the individual beingtreated. Immunosuppressive agents include, for example, non-steroidalanti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids,disease-modifying antirheumatic drugs (DMARDs) for the treatment ofarthritis, or biologic response modifiers. Compositions in the DMARDdescription are also useful in the treatment of many other autoimmunediseases aside from RA.

Exemplary NSAIDs are chosen from the group consisting of ibuprofen,naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx and Celebrex,and sialylates. Exemplary analgesics are chosen from the groupconsisting of acetaminophen, oxycodone, tramadol of proporxyphenehydrochloride. Exemplary glucocorticoids are chosen from the groupconsisting of cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, or prednisone. Exemplary biologicalresponse modifiers include molecules directed against cell surfacemarkers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNFantagonists (e.g. etanercept (Enbrel), adalimumab (Humira) andinfliximab (Remicade)), chemokine inhibitors and adhesion moleculeinhibitors. The biological response modifiers include monoclonalantibodies as well as recombinant forms of molecules. Exemplary DMARDsinclude azathioprine, cyclophosphamide, cyclosporine, methotrexate,penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold(oral (auranofin) and intramuscular) and minocycline.

It is contemplated the binding molecule composition and the furthertherapeutic agent may be given simultaneously in the same formulation.Alternatively, the agents are administered in a separate formulation butconcurrently, with concurrently referring to agents given, for example,within minutes, hours or days of each other.

In another aspect, the further therapeutic agent is administered priorto administration of the binding molecule, bifunctionalchemotherapeutic, or combination composition. Prior administrationrefers to administration of the further therapeutic agent within therange of minutes, hours, or one week prior to treatment with the bindingmolecule, bifunctional chemotherapeutic, or combination composition. Itis further contemplated that the further therapeutic agent isadministered subsequent to administration of the binding moleculecomposition. Subsequent administration is meant to describeadministration more than minutes, hours, or weeks after bindingmolecule, bifunctional chemotherapeutic, or combination compositiontreatment or administration.

It is further contemplated that when the binding molecule isadministered in combination with a further therapeutic agent, whereinthe further therapeutic agent is a cytokine or growth factor, or achemotherapeutic agent, the administration may also include use of aradiotherapeutic agent or radiation therapy. The radiation therapyadministered in combination with an antibody composition is administeredas determined by the treating physician, and at doses typically given topatients being treated for cancer.

These compositions may be administered in a single dose or in multipledoses. Standard dose-response studies, first in animal models and thenin clinical testing, reveal optimal dosages for particular diseasestates and patient populations.

The administration of the binding molecule, bifunctionalchemotherapeutic or combination composition decreases the B-cellpopulation by at least 20% after a single dose of treatment. In oneembodiment, the B-cell population is decreased by at least about 20,about 30, about 40, about 50, about 60, about 70, about 80, about 90, orabout 100%. B-cell reduction is defined as a decrease in absolute B-cellcount below the lower limit of the normal range. B-cell recovery isdefined as a return of absolute B-cell count to, for example, 70%, 80%,90% of a subject's baseline value or normal range. Further, theadministration of binding molecule, bifunctional chemotherapeutic, orcombination composition of this disclosure results in desired clinicaleffects in the disease or disorder being treated.

In some embodiments, patients suffering from a disease associated withaberrant B cell activity who receive treatment according to thedisclosure may demonstrate an overall beneficial response to thetreatment, based on clinical criteria well known and commonly used inthe art and as described below.

For example, in patients affected by rheumatoid arthritis, theadministration may improve the patient's condition by a clinicallysignificant amount [e.g., achieves the American College of RheumatologyPreliminary Detection of Improvement (ACR20)], and/or an improvement of20% in tender and swollen joint and 20% improvement in 3/5 remaining ACRmeasures (Felson et al., Arthritis Rheum. 1995, 38:727-35). Biologicalmeasures for improvement in an RA patient after administration ofCD37-specific and CD20-specific binding molecules include measurement ofchanges in cytokine levels, measured via protein or RNA levels.Cytokines of interest include, but are not limited to, TNF-α, IL-1,interferons, Blys, and APRIL. Cytokine changes may be due to reduced Bcell numbers or decreased activated T cells. In RA patients, markersrelevant to bone turnover (bone resorption or erosion) are measuredbefore and after administration of CD20-specific binding molecules.Relevant markers include, but are not limited to, alkaline phosphatase,osteocalcin, collagen breakdown fragments, hydroxyproline,tartrate-resistant acid phosphatase, and RANK ligand (RANKL). Otherreadouts relevant to the improvement of RA include measurement of Creactive protein (CRP) levels, erythrocyte sedimentation rate (ESR),rheumatoid factor, CCP (cyclic citrullinated peptide) antibodies andassessment of systemic B cell levels and lymphocyte count via flowcytometry. Specific factors can also be measured from the synovium of RApatients, including assessment of B cell levels in synovium fromsynovium biopsy, levels of RANKL and other bone factors and cytokinesset out above.

In a related aspect, the effects of combination administration on otherdiseases may be measured according to standards known in the art. Forexample, it is contemplated that Crohn's disease patients treatedaccording to the invention achieve an improvement in Crohn's DiseaseActivity Index (CDAI) in the range of about 50 to about 70 units,wherein remission is at 150 units (Simonis et al, Scand. J Gastroent.1998, 33:283-8). A score of 150 or 200 is considered normal, while ascore of 450 is considered a severe disease score. It is further desiredthat administration of the CD37-specific and CD20-specific bindingmolecules results in a reduction in perinuclear anti-neutrophil antibody(pANCA) and anti-Saccharomyces cervisiae antibody (ASCA) in individualsaffected by inflammatory bowel disease.

It is further contemplated that adult and juvenile myositis patientstreated according to the disclosure may achieve an improvement in coreset of evaluations, such as 3 out of 6 of the core set measured improvedby approximately 20%, with not more than 2 of the core measurementsworse by approximately 25% (see Rider et al., Arthritis Rheum. 2004,50:2281-90).

It is further contemplated that SLE patients treated according to thedisclosure may achieve an improvement in Systemic Lupus Activity Measure(SLAM) or SLE Disease Activity Index (SLEDAI) score of at least 1 point(Gladman et al, J Rheumatol 1994, 21:1468-71) (Tan et al., ArthritisRheum. 1982, 25:1271-7). A SLAM score of >5, or SLEDAI score>2, isconsidered clinically active disease. A response to treatment may bedefined as improvement or stabilization over the in 2 disease activitymeasures (the SLE Disease Activity Index [SLEDAI] and the Systemic LupusActivity Measure) and 2 quality of life measures (patient's globalassessment and the Krupp Fatigue Severity Scale) (Petri et al.,Arthritis Rheum. 2004, 50:2858-68.) It is further contemplated thatadministration of the binding molecule to SLE patients results in areduction in anti-double-stranded DNA antibodies. Alternatively,improvement may be gauged using the British Isles Lupus Assessment GroupCriteria (BILAG).

It is further contemplated that multiple sclerosis patients treatedaccording to the disclosure may achieve an improvement in clinical scoreon the Kurtzke Expanded Disability status scale (EDSS) (Kurtzke, F.,Neurology 1983, 33:1444-52) of at least 0.5, or a delay in worsening ofclinical disease of at least 1.0 on the Kurtzke scale (Rudick et al.,Neurology 1997, 49:358-63).

It is further contemplated that patients suffering from IIM treatedaccording to the disclosure may achieve a reduction in at least one offive criteria set out in the Idiopathic Inflammatory Myopathy Criteria(IIMC) assessment (Miller, F., supra). It is further contemplated thatadministration to IIM patients may result in a reduction in IIMassociated factors selected from the group consisting of creatine kinase(CK), lactate dehydrogenase, aldolase, C-reactive protein, aspartateaminotransferase (AST), alanine aminotransferase (ALT), and antinuclearautoantibody (ANA), myositis-specific antibodies (MSA), and antibody toextractable nuclear antigens. Alternatively, patients meet 3 out of 6 ofthe criteria set out in Rider et al., Arthritis Rheum., 50(7):2281-2290(2004), with worsening in no more than 2 criteria.

In some embodiments, patients suffering from a B cell cancer thatreceive treatment according to the disclose may demonstrate an overallbeneficial response to the treatment, based on clinical criteriawell-known and commonly used in the art, and as described below, such asa decrease in tumor size, decrease in tumor number and/or an improvementin disease symptoms.

Exemplary clinical criteria are provided by the U.S. National CancerInstitute (NCI), which has divided some of the classes of cancers intothe clinical categories of “indolent” and “aggressive” lymphomas.Indolent lymphomas include follicular cell lymphomas, separated intocytology “grades,” diffuse small lymphocytic lymphoma/chroniclymphocytic leukemia (CLL), lymphoplasmacytoid/Waldenstrom'sMacroglobulinemia, Marginal zone lymphoma and Hairy cell leukemia.Aggressive lymphomas include diffuse mixed and large cell lymphoma,Burkitt's lymphoma/diffuse small non-cleaved cell lymphoma,Lymphoblastic lymphoma, Mantle cell lymphoma and AIDS-related lymphoma.In some cases, the International Prognostic Index (IPI) is used in casesof aggressive and follicular lymphoma. Factors to consider in the IPIinclude age (<60 years of age versus >60 years of age), serum lactatedehydrogenase (levels normal versus elevated), performance status (0 or1 versus 2-4) (see definition below), disease stage (I or II versus IIIor IV), and extranodal site involvement (0 or 1 versus 2-4). Patientswith 2 or more risk factors have less than a 50% chance of relapse-freeand overall survival at 5 years.

Performance status in the aggressive IPI is defined as follows: GradeDescription: 0 Fully active, able to carry on all pre-diseaseperformance without restriction; 1 Restricted in physically strenuousactivity but ambulatory and able to carry out work of a light orsedentary nature, e.g., light house work, office work; 2 Ambulatory andcapable of all selfcare but unable to carry out any work activities, upto and about more than 50% of waking hours; 3 Capable of only limitedselfcare, confined to bed or chair more than 50% of waking hours; 4Completely disabled, unable to carry on any selfcare, totally confinedto bed or chair; and, 5 Dead. (See, The International Non-Hodgkin'sLymphoma Prognostic Factors Project. A predictive model for aggressivenon-Hodgkin's lymphoma. N. Engl. J. Med. 329:987-94, 1993.)

Typically, the grade of lymphoma is clinically assessed using thecriterion that low-grade lymphoma usually presents as a nodal diseaseand is often indolent or slow-growing. Intermediate- and high-gradedisease usually presents as a much more aggressive disease with largeextranodal bulky tumors.

The Ann Arbor classification system is also used to measure progressionof tumors, especially non-Hodgkin's lymphomas. In this system, stages I,II, III, and IV of adult NHL can be classified into A and B categoriesdepending on whether the patient has well-defined generalized symptoms(B) or not (A). The B designation is given to patients with thefollowing symptoms: unexplained loss of more than 10% body weight in the6 months prior to diagnosis, unexplained fever with temperatures above38° C. and drenching night sweats. Definitions of the stages are asfollows: Stage I-involvement of a single lymph node region or localizedinvolvement of a single extralymphatic organ or site. StageII-involvement of two or more lymph node regions on the same side of thediaphragm or localized involvement of a single associated extralymphaticorgan or site and its regional lymph nodes with or without other lymphnode regions on the same side of the diaphragm. Stage III-involvement oflymph node regions on both sides of the diaphragm, possibly accompanyinglocalized involvement of an extralymphatic organ or site, involvement ofthe spleen, or both. Stage IV-disseminated (multifocal) involvement ofone or more extralymphatic sites with or without associated lymph nodeinvolvement or isolated extralymphatic organ involvement with distant(non-regional) nodal involvement. For further details, see TheInternational Non-Hodgkin's Lymphoma Prognostic Factors Project: Apredictive model for aggressive non-Hodgkin's lymphoma, New England J.Med. (1993) 329:987-994.

In one aspect, a therapeutic effect of the methods according to thedisclosure is determined by the level of response, for example, apartial response is defined as tumor reduction to less than one-half ofits original size. A complete response is defined as total eliminationof disease confirmed by clinical or radiological evaluation. In oneembodiment, the individual receiving treatment according to theinvention demonstrates at least a partial response to treatment.

According to the Cheson criteria for assessing NHL developed incollaboration with the National Cancer Institute (Cheson et al., J ClinOncol. 1999, 17:1244; Grillo-Lopez et al., Ann Oncol. 2000, 11:399-408),a complete response is obtained when there is a complete disappearanceof all detectable clinical and radiographic evidence of disease anddisease-related symptoms, all lymph nodes have returned to normal size,the spleen has regressed in size, and the bone marrow is cleared oflymphoma.

An unconfirmed complete response is obtained when a patient showscomplete disappearance of the disease and the spleen regresses in size,but lymph nodes have regressed by more than 75% and the bone marrow isindeterminate. An unconfirmed complete response meets and exceeds thecriteria for partial response. An overall response is defined as areduction of at least 50 percent in overall tumor burden.

Similar criteria have been developed for various other forms of cancersor hyperproliferative diseases and are readily available to a person ofskill in the art. See, e.g., Cheson et al., Clin Adv Hematol Oncol.2006, 4:4-5, which describes criteria for assessing CLL; Cheson et al.,J Clin Oncol. 2003, 21:4642-9, which describes criteria for AML; Chesonet al., Blood 2000, 96:3671-4, which describes criteria formyelodysplastic syndromes.

In another aspect, a therapeutic response in patients having a B cellcancer is manifest as a slowing of disease progression compared topatients not receiving therapy. Measurement of slowed diseaseprogression or any of the above factors may be carried out usingtechniques well-known in the art, including bone scan, CT scan, galliumscan, lymphangiogram, MRI, PET scans, ultrasound, and the like.

As an additional aspect, the disclosure includes kits which comprise oneor more compounds or compositions useful in the methods of thisdisclosure packaged in a manner which facilitates their use to practicemethods of the disclosure. In a simplest embodiment, such a kit includesa compound or composition described herein as useful for practice of amethod of the disclosure packaged in a container such as a sealed bottleor vessel, with a label affixed to the container or included in thepackage that describes use of the compound or composition to practicethe method of the disclosure. Preferably, the compound or composition ispackaged in a unit dosage form. The kit may further include a devicesuitable for administering the composition according to a preferredroute of administration or for practicing a screening assay. The kit mayinclude a label that describes use of the binding moleculecomposition(s) in a method of the disclosure.

EXAMPLES Example 1 CD37-Specific Binding Molecules

Various CD37-specific binding proteins can be made with exemplarycomponents provided in Tables 2-4. For example, antibodies or SMIPmolecules can be made, and these molecules can be chimeric, humanized,or human. More specifically, preferred light chain variable region CDRsare found in SEQ ID NOS:236-240 and 247-254 and preferred heaving chainvariable domain CDRs include SEQ ID NOS:241-245 and 247-254. Also,preferred light and heavy chain variable regions are provided in SEQ IDNOS:236-240 and SEQ ID NOS:241-245, respectively. Preferred light andheavy chain variable regions may also be found in SEQ ID NOS:247-254.Preferred variable domain linkers include SEQ ID NOS:225-229, whilepreferred hinges include SEQ ID NOS:230-235.

A particularly preferred embodiment is CAS-024 [G28-1 VH (M99F,Y102S)-VL (T25A) scFv (SSC-P) H WCH2 WCH3], which is a recombinant, 483amino acid single-chain fusion protein that binds to human CD37. Thebinding domain comprises a humanized scFv based on the G28-1 antibodyvariable region CDRs, including mutations in the heavy chain CDR3 and inthe light chain CDR1. The variable domains are linked by a (G₄5)₅ (25amino acid) sequence (SEQ ID NO:229), which is connected via a threeamino acid junction (GDQ) to the amino terminus of a modified upper andcore IgG1 hinge region (wherein the first two of three cysteines foundin these hinge regions are each substituted with a serine). Thecarboxy-terminus of the hinge is fused to an effector domain comprisingCH2 and CH3 domains of IgG₁. The amino acid sequence of CAS-024 is setout in SEQ ID NO:253. FIG. 1 shows heavy and light chain variable regionamino acid sequence alignments of mouse G28.1 and CAS-024 sequences,along with a consensus identity sequence.

TABLE 1 Exemplary CD-37 Specific SMIP Constructs AA SEQ ID ConstructDescription† Linker Hinge* NO. CAS-001 Vk3:VH5-51 16aa SSC-P 6(G₄S)₂(G₄T)G CAS-002 Vk3:VH5 JH4 CDRL1 (T25A); 16aa SSC-P 48 CDRH3(M99F) (G₄S)₂(G₄T)G CAS-003 Vk3:VH5 JH5a CDRL1 (T25A); 16aa SSC-P 52CDRH3 (M99F; Y102S) (G₄S)₂(G₄T)G CAS-007 Vk3:VH5-51 (Linker TG→SS) 16aaSSC-P 8 (G₄S)₃S CAS-008 Vk3:VH5-51 VH V11S 16aa SSC-P 10 (G₄S)₂(G₄T)GCAS-009 Vk3:VH5-51 CDRL1 (E27Q) 16aa SSC-P 12 (G₄S)₂(G₄T)G CAS-010Vk3:VH5-51 CDRL1 (N28S) 16aa SSC-P 14 (G₄S)₂(G₄T)G CAS-011 Vk3:VH5-51CDRL1 (T25A) 16aa SSC-P 16 (G₄S)₂(G₄T)G CAS-012 mVk:VH5-5a 16aa SSC-P 18(G₄S)₂(G₄A)S CAS-013 Vk3:VH5 VH3 FW1 16aa SSC-P 22 (G₄S)₂(G₄A)S CAS-014mVH:Vk3 22aa SSC-P 24 (G₄S)₄AS CAS-015 Vk3:mVH (2H7 Leader) 16aa SSC-P26 (G₄S)₂(G₄A)S CAS-016 mVH:Vk3 22aa SCC-P 28 (G₄S)₄AS CAS-017 Vk3:mVH16aa SSC-P 30 (G₄S)₂(G₄A)S CAS-018 Vk3:mVH 16aa SCC-P 32 (G₄S)₂(G₄A)SCAS-019 Vk3:VH5 VH3 FW1 16aa SCC-P 34 (G₄S)₂(G₄A)S CAS-020 Vk3:VH5VH3-13 FW1 16aa SSC-P 38 (G₄S)₂(G₄A)S CAS-021 Vk3:VH5 VH3-13 FW1 16aaSCC-P 40 (G₄S)₂(G₄A)S CAS-022 Vk3:VH5 VH3-13 V11S FW1 16aa SSC-P 42(G₄S)₂(G₄A)S CAS-023 Vk3:VH5 VH3-13 V11S FW1 16aa SCC-P 44 (G₄S)₂(G₄A)SCAS-024 VHVL 25aa SSC-P 253 (G₄S)₅ CAS-060 Vk3:VH5 VH3 FW1 16aa SSC-P 36(G₄S)₂(G₄A)S CAS-061 Vk3:VH5 CDRL1 (T25A, E27Q) 16aa SSC-P 46(G₄S)₂(G₄T)G CAS-062 Vk3:CDR-H3 JH6 CDRL1 (T25A); 16aa SSC-P 254 CDRH3(Y102V) (G₄S)₂(G₄T)G CAS-063 Vk3:VH5 JH5b CDRL1 (T25A); 16aa SSC-P 266CDRH3 (M99F; Y102P) (G₄S)₂(G₄T)G CAS-064 Vk3:VH5 JH1 CDRL1 (T25A) 16aaSSC-P 267 CDRH3 (D101E; Y102H) (G₄S)₂(G₄T)G CAS-065 Vk3:CDR-H3 JH3aCDRL1 (T25A) 16aa SSC-P 268 CDRH3 (M99F; Y102V) (G₄S)₂(G₄T)G CAS-066Vk3:CDR-H3 JH3b CDRL1 (T25A) 16aa SSC-P 269 CDRH3 (M99F; Y102I)(G₄S)₂(G₄T)G CAS-067 Vk3:CDR-H3 JH2 CDRL1 (T25A) 16aa SSC-P 80 CDRH3(M99F; Y102L) (G₄S)₂(G₄T)G CAS-068 Vk3:VH5 JH2 CDRL1 (T25A) 16aa SSC-P82 CDRH2 (T59N; N61A; R62Q; K65Q) (G₄S)₂(G₄A)S CAS-069 Vk3:VH5 JH2 CDRL1(T25A) 16aa SSC-P 262 CDRH2 (T59G; N61A; R62Q; K65Q) (G₄S)₂(G₄A)SCAS-070 Vk3:VH5 JH5a CDRL1 (T25A); 20aa CPPCP 84 CDRH3 (M99F; Y102S)(G₄S)₃(G₃A)S *Entries represent abbreviations regarding IgG1 hingeshaving mutations in only the first or the first and second cysteinesfound within the upper and core regions. The only exception is SEQ IDNO: 84, which depicts the full-length hinge amino acid (CPPCP, SEQ IDNO: 230) sequence used (essentially, only the core IgG1 seqeunce with aproline at the end). †CDR mutation numbering is based on the Kabatnumbering scheme.

Additional hinge regions that may be used in CD-37 specific bindingmolecules, such as SMIP molecules or antibodies, are provided in thefollowing table.

TABLE 2 Exemplary Hinge Regions for CD37-Specific Binding Proteins HingeSEQ desHcription Amino Acid Sequence ID NO: ccc(p)-hIgG1 EPKSCDKTHTCPPCP90 scc(p)-hIgG1 EPKSSDKTHTCPPCP 92 scc(s)-hIgG1 EPKSSDKTHTCPPCS 94csc(p)-hIgG1 EPKSCDKTHTSPPCP 102 csc(s)-hIgG1 EPKSCDKTHTSPPCS 104ccs(p)-hIgG1 EPKSCDKTHTCPPSP 255 ccs(s)-hIgG1 EPKSCDKTHTCPPSS 256ssc(p)-hIgG1 EPKSSDKTHTSPPCP 106 ssc(s)-hIgG1 EPKSSDKTHTSPPCS 108scs(p)-hIgG1 EPKSSDKTHTCPPSP 257 scs(s)-hIgG1 EPKSSDKTHTCPPSS 96css(p)-hIgG1 EPKSCDKTHTSPPSP 110 css(s)-hIgG1 EPKSCDKTHTSPPSS 112sss(p)-hIgG1 EPKSSDKTHTSPPSP 98 sss(s)-hIgG1 EPKSSDKTHTSPPSS 100 hIgA1VPSTPPTPSPSTPPTPSPS 115 hIgA2 VPPPPP 116 hIgG3 ELKTPLGDTTHTCPRCPEP 118KSCDTPPPCPRCPEPKSCD TPPPCPRCPEPKSCDTPPP CPRCP hIgG3(ccc) EPKSCDTPPPCPRCP258 hIgG3(scc) EPKSSDTPPPCPRCP 120 hIgG3(csc) EPKSCDTPPPSPRCP 126hIgG3(ccs) EPKSCDTPPPCPRSP 259 hIgG3(ssc) EPKSCDTPPPSPRCP 260 hIgG3(scs)EPKSCDTPPPCPRSP 261 hIgG3(css) EPKSCDTPPPSPRSP 122 hIgG3(sss)EPKSSDTPPPSPRSP 124 hIgD ESPKAQASSVPTAQPQAEG 127 SLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKT P

Additional framework regions that may be used in CD-37 specific bindingmolecules, such as SMIP molecules or antibodies, are provided in thefollowing tables.

TABLE 3A Human Heavy Chain Framework Regions forCD37-Specific Binding Proteins SEQ V-region Human VH Framework RegionsID NO. FR1 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 140 VH1QVQLVQSGAEVKKPGSSVKVSCKASGGTFS 141 VH1 EVQLVQSGAEVKKPGATVKISCKVSGYTFT143 VH5 EVQLVQSGAEVKKPGESLKISCKGSGYSFT 144 VH5EVQLVQSGAEVKKPGESLRISCKGSGYSFT 145 VH7 QVQLVQSGSELKKPGASVKVSCKASGYTFT146 FR2 VH1 WVRQAPGQGLEWMG 147 VH1 WVQQAPGKGLEWMG 150 VH5 WVRQMPGKGLEWMG151 FR3 VH1 RVTMTTDTSTSTAYMELRSLRSDDTAVYYC 154 AR VH1RVTITADESTSTAYMELSSLRSEDTAVYYC 155 AR VH1 RVTITADKSTSTAYMELSSLRSEDTAVYYC156 AR VH1 RVTITADTSTDTAYMELSSLRSEDTAVYYC 157 AT VH5QVTISADKSISTAYLQWSSLKASDTAMYYC 158 AR VH5 HVTISADKSISTAYLQWSSLKASDTAMYYC159 AR VH7 RFVFSLDTSVSTAYLQISSLKAEDTAVYYC 160 AR FR4 JH1, JH4,WGQGTLVTVSS 161 JH5a, JH5b JH2 WGRGTLVTVSS 162 JH3a, JH3b WGQGTMVTVSS163 JH6 WGQGTTVTVSS 168 WGKGTTVTVSS 169

TABLE 3B Human Light Chain Framework Regions forCD37-Specific Binding Proteins SEQ V-region Human VK Framework RegionsID NO. FR1 VK3 EIVMTQSPATLSVSPGERATLSC 170 VK3 EIVLTQSPATLSLSPGERATLSCS171 VK1 DIQMTQSPSSLSASVGDRVTITC 172 VK1 NIQMTQSPSAMSASVGDRVTITC 175 VK1AIQLTQSPSSLSASVGDRVTITC 177 VK1 DIQLTQSPSFLSASVGDRVTITC 178 VK1AIRMTQSPFSLSASVGDRVTITC 179 VK1 AIQMTQSPSSLSASVGDRVTITC 180 VK1DIQMTQSPSTLSASVGDRVTITC 181 FR2 VK3 WYQQKPGQAPRLLIY 182 VK1WYQQKPGKAPKLLIY 184 VK1 WYQQKPGKVPKLLIY 185 VK1 WYQQKPGKAPKRLIY 186 VK1WFQQKPGKVPKHLIY 187 VK1 WFQQKPGKAPKSLIY 188 VK1 WYQQKPAKAPKLFIY 191 FR3VK3 GIPARFSGSGSGTEFTLTISSLQSED 194 FAVYYC VK3 GIPARFSGSGSGTDFTLTISSLEPED195 FAVYYC VK1 GVPSRFSGSGSGTDFTLTISSLQPED 196 FATYYC VK1GVPSRFSGSGSGTDFTLTISSLQPED 197 VATYYC VK1 GVPSRFSGSGSGTEFTLTISSLQPED 198FATYYC VK1 GVPSRFSGSGSGTDYTLTISSLQPED 203 FATYYC VK1GVPSRFSGSGSGTEFTLTISSLQPDD 205 FATYYC FR4 JK1 FGQGTKVEIK 206 FGQGTKLEIK207 FGPGTKVDIK 208 FGGGTKVEIK 209 FGQGTRLEIK 210

Preferred exemplary component parts of CD-37 specific SMIP molecules(including leader sequences used for expression and export, but whichare removed from the mature fusion protein when exported from a cell;linker sequences used to join light and heavy chain variable domains toform scFv binding domains; hinges used to join scFv binding domains toeffector domains; and effector domains), as well as certain CD-37specific SMIP molecules, including the preferred CAS-024 fusion protein,are provided in Table 4.

TABLE 4 SMIP Component Parts and Select CD37-Specific SMIP PolypeptidesSEQ Construct No. ID NO. Amino Acid Sequence Leader Sequence 223MDFQVQIFSFLLISASVIIARGV Leader Sequence 224 MEAPAQLLFLLLLWLPDTTGVariable Domain 225 GGGGSGGGGSGGGGSS Linker Variable Domain 226GGGGSGGGGSGGGGAS Linker Variable Domain 227 GGGGSGGSGSGGGGAS LinkerVariable Domain 228 GGGGSGGGGSGGGGTG Linker Variable Domain 229GGGGSGGGGSGGGGSGGGGSGGGGS Linker Hinge 230 CPPCP Hinge (junction 231SEPKSSDKTHTSPPCP amino acid italicized) Hinge (junction 232DLEPKSSDKTHTSPPCP amino acid italicized) Hinge (junction 233DQEPKSSDKTHTSPPCP amino acid italicized) Hinge (junction 234GDQEPKSSDKTHTSPPCP amino acid italicized) Hinge (junction 235GSSEPKSSDKTHTSPPCP amino acid italicized) Mouse CD37 VL 236DIQMTQSPASLSASVGETVTITC

WYQQKQ (CDRs highlighted) GKSPQLLVS

GVPSRFSGSGSGTQFSLKISSLQPE DSGSYFC

FGGGTELEIK Humanized CD37 VL 237 EIVLTQSPATLSLSPGERATLSC

WYQQKPG (CDRs highlighted)a QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIK Humanized CD37 VL 238 EIVLTQSPATLSLSPGERATLSC

WYQQKPG (CDRs highlighted)b QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIK Humanized CD37 VL 239 EIVLTQSPATLSLSPGERATLSC

WYQQKPG (CDRs highlighted)c QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIK Humanized CD37 VL 240 EIVLTQSPATLSLSPGERATLSC

WYQQKPG (CDRs highlighted)d QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIK Mouse CD37 VH 241 AVQLQQSGPESEKPGASVKISCKASGYSFT

WVKQ (CDRs highlighted) NNGKSLEWIG

KATLTVDKSSSTA YMQLKSLTSEDSAVYYCAR

WGQGTSVTVSS Humanized CD37 VH 242 EVQLVQSGAEVKKPGESLKISCKGSGYSFT

WVRQ (CDRs highlighted)a MPGKGLEWMG

QVTISADKSISTA YLQWSSLKASDTAMYYCAR

WGQGTLVTVSS Humanized CD37 VH 243 EVQLVQSGAEVKKPGESLKISCKGSGYSFT

WVRQ (CDRs highlighted)a MPGKGLEWMG

QVTISADKSISTA YLQWSSLKASDTAMYYCAR

WGQGTLVTVSS Humanized CD37 VH 244 EVQLVQSGAEVKKP[GESLKISCKGSGYSFT

WVRQ (CDRs highlighted)b MPGKGLEWMG

QVTISADKSISTA YLQWSSLKASDTAMYYCAR

WGQGTLVTVSS Humanized CD37 VH 245 EVQLVQSGAEVKKPGESLKISCKGSGYSFT

WVRQ (CDRs highlighted)c MPGKGLEWMG

QVTISADKSISTA YLQWSSLKASDTAMYYCAR

WGQGTLVTVSS IgG1 CH2CH3 246 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK CAS-006 247DIQMTQSPASLSASVGETVTIT

WYQQKQ (chimeric anti-CD37 SMIP) GKSPQLLVS

GVPSRFSGSGSGTQFSLKISSLQPE DSGSYFC

FGGGTELEIKGGGGSGGGGSGGG GSSAVQLQQSGPESEKPGASVKISCKASGYSFT

WV KQNNGKSLEWIG

KATLTVDKSSS TAYMQLKSLTSEDSAVYYCAR

WGQGTSVTVS S DL EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CAS-001 248 EIVLTQSPATLSLSPGERATLSC

WYQQKPG QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIKGGGGSGGGGSGGGG TGEVQLVQSGAEVKKPGESLKISCKGSGYSFT

WV RQMPGKGLEWMG

QVTISADKSI STAYLQWSSLKASDTAMYYCAR

WGRGTLVTV SS DQ EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CAS-002 249EIVLTQSPATLSLSPGERATLSC

WYQQKPG QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIKGGGGSGGGGSGGGG TGEVQLVQSGAEVKKPGESLKISCKGSGYSFT

WV RQMPGKGLEWMG

QVTISADKSI STAYLQWSSLKASDTAMYYCAR

WGQGTLVTV SS DQ EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK CAS-003 250 EIVLTQSPATLSLSPGERATLSC

WYQQKPG QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIKGGGGSGGGGSGGGG TGEVQLVQSGAEVKKPGESLKISCKGSGYSFT

WV RQMPGKGLEWMG

QVTISADKSI STAYLQWSSLKASDTAMYYCAR

WGQGTLVTV SS DQ EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CAS-014 251AVQLQQSGPESEKPGASVKISCKASGYSFT

WVKQ (mouse-human hybrid) NNGKGLEWIG

KATLTVDKSSSTA YMQLKSLTSEDSAVYYCAR

WGQGTSVTVSSG GGGSGGGGSGGGGSGGGGSASEIVLTQSPATLSLSPGERAT LSC

WYQQKPGQAPRLLIY

GIPA RFSGSGSGTDFTLTISSLEPEDFAVYYC

FGQ GTKVEIK GSS EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK CAS-017 252EIVLTQSPATLSLSPGERATLSC

WYQQKPG (human-mouse hybrid) QAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDF AVYYC

FGQGTKVEIKGGGGSGGGGSGGGG ASAVQLQQSGPESEKPGASVKISCKASGYSFT

WV KQNNGKGLEWIG

KATLTVDKSSS TAYMQLKSLTSEDSAVYYCAR

WGQGTSVTVS S S EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK CAS-024 253EVQLVQSGAEVKKPGELSKISCKGSGYSFT

WVRQ MPGKGLEWMG

QVTISADKSISTA YLQWSSLKASDTAMYYCAR

WGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPG ERATLSC

WYQQKPGQAPRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC

FGQGTKVEIK GDQ EPKSSDKTHTSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

Example 2 Expression of CAS-024 and other CD37-Specific Binding Proteins

CAS-024 and other CD37-specific binding SMIP molecules were cloned intoa Chinese Hamster Ovary (CHO) mammalian cell expression system.Transfected CHO cells that produced the SMIP molecules were cultured inshake flasks and harvested cell culture supernatants were titered usingOctec Q Protein A sensor.

Table 5 shows that the CAS-024 construct (VHVL format with 25 amino acidvariable domain linker) had an unexpectedly superior expression level,up to about 10-fold better, than the other humanized anti-CD37 SMIPmolecules (mostly VLVH format with 15 amino acid variable domainlinker). Indeed, all fully humanized VLVH constructs expressed poorly(data not shown), as did mouse-human hybrid molecules in eitherorientation (see Example 5).

TABLE 5 SMIP Expression SMIP Protein Clones Screened Protein Titer Range(μg/ml) CAS-001 492 65-80 CAS-002 425 200-280 CAS-003 611 300-360CAS-024 203 500-650

Example 3 Purification and Size Exclusion Chromatography of CAS-024 andOther CD37-Specific Binding Proteins

To produce more protein, nucleic acid encoding CAS-024 and several otherCD37-specific binding SMIP molecules were cloned into a Chinese HamsterOvary (CHO) mammalian cell expression system. Transfected CHO cells thatproduced the SMIP molecules were cultured in shake flasks.

All of the CD37-specific binding SMIP molecules were purified from CHOculture supernatants by Protein A affinity chromatography. A 50 mLrProtein A FF sepharose column (GE Healthcare) was equilibrated at 5.0mls/min (150 cm/hr) for 1.5 column volumes (CV) with dPBS. The culturesupernatant was loaded onto the rProtein A Sepharose FF column at a flowrate of 1.7 mls/min using the AKTA Explorer 100 Air (GE healthcare),capturing the recombinant SMIP molecules. The column was washed withdPBS for 5 Column Volumes (CV), then 1.0 M NaCl, 20 mM Sodium Phosphate,pH 6.0, and then with 25 mM NaCl, 25 mM NaOAc, pH 5.0. The recombinantCD37-specific binding molecules were eluted from the column with 100 mMglycine, pH 3.5. Fractions (10 mL) of the eluted product were recoveredand then brought to pH 5.0 with 20% of the eluted volume of 0.5 M2-(N-morpholino)ethanesulfonic acid (MES), pH6.0. This eluted productwas concentrated to approximately 25 mg/mL protein and filtersterilized.

This concentrated and sterilized protein was further purified by GPCsize exclusion chromatography (SEC) to achieve separate SMIP (dimer)molecule from higher molecular weight aggregates. An XK 50/100 column(GE healthcare) containing 1 L of Superdex 200 FF sepharose wasequilibrated at 12.6 ml/min (38 cm/hr) for 1.5 column volumes (CV) withdPBS. A maximum volume of 54 mls (3% CV) of sample was applied to thecolumn. The column continued to run at 12.6 ml/min and the elutedprotein was fractionated in 40 mL fractions. Each fraction was analyzedfor product quality using an analytic HPLC, and the eluted fractionswere pooled to greater than about 95% protein of interest(non-aggregated). The resultant pool was filter sterilized at 0.22 μm,concentrated, and then formulated with 20 mM sodium phosphate, 240 mMsucrose, pH 6.0.

The SEC traces showing the peaks containing the protein of interest(POI) for CAS-001 (SEQ ID NO:6), CAS-002 (SEQ ID NO:48), CAS-003 (SEQ IDNO:52), and CAS-024 (SEQ ID NO:253) are shown in FIGS. 2A-2D,respectively. The CAS-024 peak is narrower and more symmetric than theCAS-001, CAS-002, and CAS-003 samples (broader and asymmetric). TheCAS-006 (chimeric) molecule produces a sharp peak similar to CAS-024.The CAS-001, CAS-002, and CAS-003 samples all had a slight tailingshoulder, which if integrated, accounts for about 35% of the POI area.This ‘shoulder’ would be difficult to separate from the POI and probablyrepresents either misfolded conformers or a heterogenous population ofmolecules (e.g., have different levels of glycosylation). This indicatesthat CAS-024 was not only expressed better, but this construct alsoproduces a more homogenous population of molecules.

Example 4 Cell Binding by CAS-024 is Unexpectedly Superior to OtherCD37-Specific Binding Proteins

A competition assay was used to compare the binding affinity ofdifferent anti-CD37 specific small modular immunopharmaceutical (SMIP)molecules to CD-37 found on Ramos cells (a B-lymphoblastoid cell linederived from a Burkitt lymphoma). An SEC purified chimeric anti-CD37SMIP molecule (CAS-006, SEQ ID NO:247) was labeled with the FMAT Blue®fluorescence dye (Applied Biosystems) and used as the standard tocompete with purified unlabeled chimeric anti-CD37 SMIP molecule(positive control) and purified unlabeled humanized anti-CD37 SMIP testmolecules. Higher affinity showed up as a weaker fluorescence signal andan FL1 fluorescence value was used to generate the competition curve.Briefly, the reagent FMAT Blue® labeled chimeric anti-CD37 SMIP moleculewas diluted to 2 μg/ml in FACS blocking buffer and the purified proteinsamples (CAS-001 (SEQ ID NO:6), CAS-002 (SEQ ID NO:48), CAS-003 (SEQ IDNO:52), and CAS-024 (SEQ ID NO:253)) were serially diluted 1:2 toconcentrations ranging from 50 μg/ml to 0.02 μg/ml. Ramos cells wereharvested at 1,000 rpm for 5 minutes and resuspend in FACS blockingbuffer at 4×10⁶ cells/10 ml buffer. To each well of a black 96-wellplate the following was added: 50 μl sample, 50 μl FMAT Blue® labeledchimeric anti-CD37 SMIP molecule, and 50 μl Ramos cells (4×10⁴/well).The plates were incubated at room temperature for 30 min and read on an8200 Cellular Detection System (Applied Biosystems) gated for middlecell size and low signal.

This FMAT competition assay showed that CAS-024 (humanized anti-CD37SMIP molecule having a VHVL scFv with a 25 amino acid variable domainlinker) has the same affinity for CD37 as the parent chimeric anti-CD37SMIP molecule and, in contrast, has an unexpectedly up to a 4-foldgreater affinity for CD37 than the humanized anti-CD37 SMIP moleculeshaving the reverse VLVH structure and a shorter 16 amino acid variabledomain linker (see FIG. 3). The best binding, although stillsignificantly less than CAS-006 or CAS-024, was found in a VLVHconstruct that does not have any CDR mutations (CAS-001). However,CAS-001 was consistently the worst expressing construct and produced anon-homogenous population of purified molecules—even for this construct,CAS-024 bound from 1.5- to 2-fold better than CAS-001.

This result was also surprising because M99 and Y102 in the heavy chainCDR3 of CAS-024 are mutated—the Y102 position is generally conserved andit would be expected that a change at this position alone would diminishor even abolish binding (e.g., CAS-062, mutated at postion Y102, hasdetectable but severely decreased binding compared to CAS-001 orCAS-024, while CAS-063 to CAS-067 each have barely detectable to nobinding activity in this assay when a mutation at postion M99 or D101 isadded, data not shown). Thus, the structure of CAS-024 provided amolecule that surprisingly bound as well as the chimeric molecule,CAS-006.

Example 5 Expression and Cell Binding of CAS-024 Compared to Mouse-HumanHybrid CD37-Specific Binding Proteins

CAS-024 and other CD37-specific binding SMIP molecules were produced byrecombinant DNA technology and transfected into HEK293 cells for 7 days.Cell culture supernatants were harvested on day 7 and titered usingOctec Q Protein A sensor.

Similar to the results found in Example 2, here Table 6 shows thatCAS-024 (VHVL format with 25 amino acid variable domain linker)expressed from about 5-fold to about 27-fold better than the otherhumanized or mouse-human hybrid anti-CD37 SMIP molecules. Themouse-human hybrid molecules did not express well regardless of the VHVLor VLVH orientation.

TABLE 6 SMIP Expression SMIP Protein Protein Titer (μg/ml) CAS-002(hVLhVH) 0.47 CAS-003 (hVLhVH) 2.39 CAS-014 (mVHhVL) 2.16 CAS-017(hVLmVH) 0.70 CAS-006 (mVLmVH) 9.3 CAS-024 (hVHhVL) 12.7

A competition assay as described in Example 4 was used to compare thebinding affinity of different mouse-human hybrid anti-CD37 SMIPmolecules compared to CAS-024 binding to Ramos cells. An SEC purifiedchimeric anti-CD37 SMIP molecule (CAS-006, SEQ ID NO:247) was labeledwith the FMAT Blue® fluorescence dye (Applied Biosystems) and used asthe standard to compete with purified unlabeled chimeric anti-CD37 SMIPmolecule (CAS-006, positive control) and purified unlabeled humanizedanti-CD37 SMIP test molecules—CAS-002 (SEQ ID NO:48), CAS-003 (SEQ IDNO:52), CAS-014 (SEQ ID NO:251), CAS-017 (SEQ ID NO:252), and CAS-024(SEQ ID NO:253).

This FMAT competition assay again showed that CAS-024 (VHVL humanizedmolecule with a 25 amino acid linker) has the same affinity for CD37 asthe parent chimeric anti-CD37 SMIP molecule (CAS-006), whereas CAS-002and CAS-003 (VLVH humanized molecules with a 16 amino acid linker) didnot bind as well (showing a 2- to 3-fold reduction) (see FIG. 4A). Themouse-human hybrid molecules, regardless of variable domain orientation(mouseVH-humanizedVL with a 22 amino acid linker or humanizedVL-mouseVHwith a 16 amino acid linker), bound as well as or even better than (1.5-to 2-fold) CAS-006 and CAS-024 (see FIG. 4B). These data show that amouse-human hybrid molecule without mutations binds as well as or betterthan CAS-006, regardless of orientation, and that a fully humanized VLVHconstruct with no CDR mutations binds better than other humanizedmolecules but still has diminished binding compared to CAS-006 orCAS-024. Together, these data suggest that no mutations in CDRs for thismolecule would be better binders. Also, the particular order (VLVH orVHVL) did not seem to solve the expression problem, even when a longervariable domain linker is used (see CAS-014). Thus, it was unpredictableto select a molecule with the CAS-024 structure and properties similarto the parent molecule CAS-006.

Example 6 CAS-006 and Various CD37-Specific Antibodies Bind Same or anOverlapping Epitope on CD37

Experiments were performed to identify the CD37 epitope bound by CAS-006and other previously described CD37-specific antibodies. UnconjugatedMB371 (#555457) and FITC-conjugated MB371 (#555456) were obtained fromBD Pharmingen (San Jose, Calif.), FITC-conjugated BL14 (#0457) fromImmunotech/Beckman Coulter (Fullerton, Calif.), FITC-conjugated NMN46(#RDI-CBL 136FT) and unconjugated NMN46 (#RDI-CBL 136) from RDI(Flanders, N.J.), FITC-conjugated IPO24 (#186-040) and unconjugatedIPO-24 (#186-020) from Ancell Corporation (Bayport, Minn.),FITC-conjugated HHI (#3081) and unconjugated HH1 (#3080) from DiaTec.Com(Oslo, Norway) and FITC-conjugated WR17 (YSRTMCA483F) and unconjugatedWR17 (YSRTMCA483S) from Accurate Chemical & Scientific (Westbury, N.Y.).CAS-006 SMIP protein was produced as described in Example 2.

CAS-006 was conjugated to FITC using a Molecular Probes FluororeporterFITC Labeling Kit (F6434) according to manufacturer's instructions asfollows: CAS-006 protein peak of interest (POI) at 13.5 mg/mL wasadjusted to 5 mg/mL with PBS. One mg (200 ul) was added to kit tubeswith a stirbar, and 1M NaHCO₃ (adjusted to pH 8.5 with 6N NaOH), wasadded to a final concentration of 0.1 M. 50 ul DMSO was added to 370 ugof FITC and was added to the tubes at molar ratios of 15, 20, 30 and 40FITC: protein using the following formula to determine the ul of FITC toadd: [μl of FITC solution to add=5 mg/mL protein×0.2 mL×389×100×desiredmolar ratio/Molecular weight of CAS-006 (110,000)].

Reactions were shielded from light and stirred continuously for 75minutes at room temperature. Reactions were added to spin columnsprepared as described in the kit and spun at 1100 g for 5 minutes tobuffer exchange into PBS with azide and remove unconjugated FITC. The ODat 280 nM and 494 nM was determined with 2 ul drops on the Nanodrop; theextinction coefficient for CAS-016 was experimentally determined forthis instrument by reading dilutions of the starting unconjugated SMIPmolecule, the concentration of each of the conjugates was 4.25 mg/ml andthe following FITC:protein rations were determined: 2.7 FITC/CAS-016 ata ratio of 15; 3.7 FITC/CAS-016 at a ratio of 20; 4.4 FITC/CAS-016 at aratio of 30; and 5.1 FITC/CAS-016 at a ratio of 40.

BSA was added to 3 mg/mL to help stabilize the protein. Binding of eachfraction was assessed at dilutions ranging from 100-24,300× on Ramos and3200-25,600 on human PBMC. All bound, but the MR30 ratio was chosen forfurther use since it gave a high MFI that was well maintained over thetitration range used, indicating that binding avidity was least affectedin this reaction.

FITC labeled antibody conjugates were titrated from 10 ng/mL to 10 μg/mLin an initial binding study to determine the optimal amount to use inthe blocking studies. The level chosen was just below saturatingamounts, and was kept constant in the subsequent assays, while levels ofblocking antibody were increased over a 10-fold range. Data were plottedas percent of maximal binding versus concentration of blocking antibody,so that higher levels indicate less efficient blocking, while lowerlevels indicate more efficient blocking activity. All of the antibodiestested showed blocking activity of the maximal binding observed withoutunlabeled reagents (FIG. 5).

BJAB-cells, a lymphoblastoid B-cell line were then stained with a panelof various clones of anti-CD37 mAbs, including MB371, BL14, NMN46,IPO24, HH1, WR17, and chimeric CAS-006 SMIP.

For competitive binding assays, 2.5×10⁵ BJAB cells were incubated in96-well V-bottom plates in staining media (PBS with 2% mouse sera) withthe FITC-conjugated anti-CD37 mAbs at 1.25 μg/mL in the presence ofunconjugated anti-CD37 MAb at the indicated concentrations (2.5, 1.25,0.6, or 0.3 μg/ml) or staining media for 45 minutes on ice in the dark.Blocking antibodies and FITC labeled antibody conjugates were added toreactions prior to addition of cells. The cells were then washed 2.5times with PBS and fixed with 1% paraformaldehyde (USB, Cleveland,Ohio). The treated cells were analyzed by flow cytometry using aFACsCalibur instrument and CellQuest software (BD Biosciences, San Jose,Calif.).

For FACs cross blocking assays, 2.5×10⁵ BJAB cells were incubated in96-well V-bottom plates in staining media (PBS with 2% mouse sera) inthe presence of unconjugated anti-CD37 MAb at 5 μg/mL staining media for45 minutes at room temperature in the dark. FITC-conjugated anti-CD37mAbs were added to a final concentration of 2 μg/ml, resulting in adilution of the unlabelled reagents to 3.3 μg/ml. The reactions werefurther incubated for 45 minutes at room temperature in the dark, thenwashed 2.5 times with PBS, and finally fixed in 1% paraformaldehyde inPBS (USB, Cleveland, Ohio). Cells were analyzed by flow cytometry on aFACsCalibur instrument using Cell Quest software (BD Biosciences, SanJose, Calif.).

For cell binding assays, cells were suspended in PBS (Gibco/Invitrogen,Grand Island N.Y.) containing 2% FBS (Gibco/Invitrogen), (stainingmedia) at a concentration of approximately 4×10⁶ cells/mL. Cells werethen plated and test samples, diluted in staining media, were then added1:1 to the final designated concentrations. Reactions were incubated for45 minutes on ice. Samples were centrifuged and washed 2 times with PBS.FITC goat anti-human IgG (CalTag, Burlingame Calif.) was added at afinal dilution of 1:50, and incubated 45 minutes on ice. Samples werecentrifuged, washed in PBS, then fixed in 200 μl 1% paraformaldehyde inPBS (USB, Cleveland, Ohio). Cells were analyzed by flow cytometry on aFACsCalibur instrument using Cell Quest software (BD Biosciences, SanJose, Calif.).

Each antibody showed dose dependent inhibition of binding, indicatingthat all the molecules tested bind to an identical or closely relatedepitope. A different potency for inhibition of binding was observed foreach antibody. CAS-006 SMIP had the highest level of blocking activityof all molecules tested, while HH1 gave an intermediate level ofblocking activity, and WR17, IPO24 blocked better than MB371, but showedless effective blocking than the other two unlabeled molecules (FIG. 5).

In addition to analysis of blocking activity, a similar series ofexperiments was performed in which various CD37 targeted antibodies weretested for their ability to compete with one another for binding to theCD37 receptor. The results from these experiments, like results obtainedin the blocking studies for all the molecules tested, indicated that thevarious CD37 targeted antibodies and CAS-006 have the same or closelyoverlapping epitopes.

Example 7 Dose Response of CAS-024 in an Established Subcutaneous HumanTumor (DOHH2) Xenograft Model in SCID Mice

The objective of this experiment was to examine the dose response totreatment with CAS-024 in a model of established subcutaneous humantumor (DOHH2) xenograft model in SCID mice. DOHH2 is a CD20⁺CD37⁺ humanB-lymphoblastoid cell line derived from a patient with follicularlymphoma (Kluin-Nelemans et al., Leukemia 5:221, 1991). Thus, DOHH2 wasderived from a patient with a non-Burkitt's NHL.

Five million DOHH2 cells were injected subcutaneously into the flank offemale CB-17SCID mice (Harlan, Somerville, N.J.) at 6.5 weeks of age andat a mean weight of 18.0±0.1 g (ranging from 14.6 to 22.6 g). On day 8post-tumor inoculation, palpable tumors were apparent in a majority ofmice. The tumor-bearing mice were sorted into four groups withequivalent mean tumor volumes (n=14 per group; 2 cages of 5 mice and 1cage of 4 mice for each group). The day of the sort was defined as day0. Tumor diameters were determined with a pair of calipers and tumorvolumes were calculated using the formula: V=½[length×(width)²]. Thebaseline mean tumor volume was 228 mm³, the median baseline tumor sizewas 224 mm³, and the range was 179 to 284 mm³.

TABLE 7 Reagents for In Vivo Use % Concentration and Preparation forReagent POI Endotoxin Injection PBS NA 1X NA Endotoxin <0.03 EU/mg HumanIgG Not  10 mg/mL Diluted to 1.0 mg/mL (huIgG) tested Endotoxin = 10EU/mg PBS CAS-024 100 9.6 mg/mL Diluted to 1.0 mg/mL Endotoxin = 0.01EU/mg PBS for 200 μg dose; then diluted 1:2 to prepare 100 μg dose, thenserially diluted 1:3 to prepare the other dose solutions.

Tumor-bearing groups of SCID mice were treated on days 0, 4, and 8 viaIP injection of 0.2 mL of PBS containing 200 μg of huIgG (negativecontrol) or 200, 100, 30, 10, or 3 μg of CAS-024. The two lowest dosesolutions of CAS-024 were prepared on the day of injection to avoid theneed to add a carrier protein to the most dilute solutions. Drugsolutions were color-coded as described below (see Table 8 below).

TABLE 8 Experimental Design No. Mice, Route of Dose per CumulativeInjection, and injection mg/kg per Cumulative Dose Group ID TreatmentDays (μg) Injection^(a) Dose (μg) (~mg/kg)^(a) huIgG 14 per group 20011.1 600 33 CAS-024 IP injection 200 11.1 600 33 200 Days 0, 4, 8CAS-024 100 5.6 300 16.7 100 CAS-024 30 1.7 90 5.0 30 CAS-024 10 0.6 301.7 10 CAS-024 3 3 0.2 9 0.5 ^(a)Note that huIgG and CAS-024 weredelivered in μg per mouse, not in mg/kg. The approximate mg/kg is notedfor convenience, and is based on the mean weight (18.0 ± 0.1 g) of miceon day 0. The weight range in this experiment was 14.6 to 22.6 g.

Dose solutions were prepared in similar volumes and the contents of thetubes were noted on removable labels. An investigator who was nottreating or assessing the mice placed a color code on each tube andnoted the code and identity of the tube contents in a laboratorynotebook. Mice were monitored daily by visual inspection. Weights weredetermined weekly, and tumor diameters were determined at least 3 timesper week (M, W, F) by an observer blinded (see above) to the treatmentgroups. Tumor volumes were calculated as described above. Mice wereeuthanized if their tumor volume reached more than 1500 mm³ (or 1200 mm³on Fridays). Death was not an endpoint in the tumor protocols and,unless noted otherwise, “survival” of a mouse was determined by the timeit was euthanized due to its tumor volume reaching the predeterminedlimits. (The protocol called for mice to be euthanized if (1) theirtumor volume exceeded the parameters noted above, (2) ulceration of atumor occurred, (3) the tumor inhibited the mobility of the mouse, and(4) weight loss exceeded 20% of body weight.)

One mouse in the CAS-024 100 μg treatment group was euthanized on day 35due to weight loss>20%. This mouse had a tumor volume of 266 mm³ at thattime, and was treated as censored data for the survival analysis (noteuthanized as of day 35 due to tumor growth). For the calculation oftumor-free incidence at the end of the study, this mouse was classifiedas one that was euthanized during the study due to growth of its tumor(its tumor was growing back at the time of its death). No other micewere found dead and none were euthanized due to weight loss, tumorulceration, or impaired mobility. No overt signs of toxicity or weightloss were observed in any of the treatment groups (data not shown).

All statistical analyses were performed using GraphPad Prism software.Significant differences in mean tumor volumes and mean relative tumorvolumes were determined using a one-way ANOVA for nonparametric data(Kruskal-Wallis test) with Dunn's multiple comparison post test. Toexamine differences between each of the CAS-024 treated groups and thehuIgG group, all groups were compared. For comparisons between theCAS-024 groups only, the huIgG group was excluded. In addition, the highand middle dose (200, 100, and 30 μg) groups were analyzed as a one dataset, and the middle and low dose (30, 10, and 3 μg) groups were analyzedas another data set. Significant differences in survival of mice overtime were determined using Kaplan-Meier survival analysis with alog-rank test for comparing survival curves. Significant differences inthe incidence of tumor-free mice were determined using Fisher's exacttest. p values<0.05 were considered significant.

CAS-024 had a dose-dependent inhibitory effect on the growth of DOHH2tumors. With the exception of the low (3 μg) dose regimen group, themean tumor volume of each CAS-024 treated group was significantly lowerthan that of the human IgG treated group as early as day 5, and remainedlower through day 12. The huIgG treated mice were euthanized starting onday 12; therefore, comparisons of tumor volumes of the CAS-024 treatedgroups to the huIgG group were not performed for later time points. Interms of a dose response, there was no significant difference in themean tumor volumes of the two highest dose groups at any point in thestudy. In contrast, the mean tumor volumes of these two groups differedsignificantly from those of each of the three lower dose groups fromdays 12 through 16 (day 16 was the last evaluable timepoint for the lowdose group). Similarly, the mean tumor volumes in mice of the 30 pg and10 μg dose groups differed from each other and from the low dose groupover this same period.

The tumors in the mice treated with huIgG grew rapidly, and all of themice in this group were euthanized by day 19. As summarized in Tables 9and 10 below, the survival of mice treated with any of the CAS-024 doseregimens was prolonged relative to the huIgG treated group (p<0.0001 inall cases). In terms of a dose response, there was no significantdifference in the survival curves of mice treated with the highest (200and 100 μg) dose regimens (p=0.7091). With the exception of this groupcomparison, there was a significant difference between the survivalcurve of each dose group and the survival curve of each of the groupstreated with a lower dose regimen (p values ranged from 0.0132 to<0.0001).

TABLE 9 Median Survival Time and Incidence of Tumor-Free Mice p Valuefor Tumor- Fischer's Death (Not Free Exact Test Median Due to LargeIncidence (comparison Treatment Cumulative Survival Tumor at End of oftumor-free Group^(a) Dose Time (Days)^(b) Volume) Study^(c)incidence)^(d) HuIgG 600 μg 14 0/14 0/14 (0%) NA 200 CAS-024 600 μg  Undefined ^(ef) 0/14   11/14 (79%) ^(g) <0.0001 200 CAS-024 300 μgUndefined   1/14 ^(h) 11/14 (79%) <0.0001 100 CAS-024  90 μg 35 0/14 5/14 (36%) 0.0407 30 CAS-024  30 μg 28 0/14 0/14 (0%) NA 10 CAS-024 3 9 μg 19 0/14 0/14 (0%) NA ^(a)Mice were treated with the indicatedprotein via IP injection on days 0, 4, and 8. The numbers indicate theamount of protein (μg) injected per day. ^(b)“Survival” of a mouse wasdetermined by the day it was euthanized due to tumor growth. One mousein the CAS-024 100 μg dose group was euthanized on day 35 due to >20%weight loss. The mouse had a tumor volume of 266 mm³ at that time, andwas treated as censored data (tumor volume did not reach predeterminedlimit by day 35) for the Kaplan Meier analysis. No other mice wereeuthanized for reasons other than its tumor volume reaching thepredetermined limit. ^(c)“Tumor-free” mice had no palpable SC tumors.The absence of tumor cells was not confirmed by histology. The studyended on day 61. ^(d)Each group was compared with the HuIgG treatedcontrol group. ^(e)The median survival time is undefined when >50% ofthe mice are alive at the end of the observation period. ^(f)Values inbold face indicate that the survival curves of the indicated group aresignificantly different from those of HuIgG control (p < 0.0001 in eachcase, log rank test). ^(g)Values in bold face are significantlydifferent from the huIgG treated control group. ^(h)One mouse waseuthanized on day 35 due to >20% weight loss. The mouse had a tumorvolume of 266 mm³ at that time and was treated as censored data for theKaplan Meier analysis.

TABLE 10 p-Values for Comparison of Survival Curves and Tumor-FreeIncidence Between CAS-024 Treated Groups p Values for IndicatedComparisons Log rank test Fisher's exact test (comparison of survival(comparison of tumor- Group Comparison^(a) curves) free incidence)  200vs 100 0.7091 1.0000 200 vs 30 0.0132 ^(b) 0.0542 200 vs 10 <0.0001<0.0001 200 vs 3  <0.0001 <0.0001 100 vs 30 0.0035 0.0542 100 vs 10<0.0001 <0.0001 100 vs 3  <0.0001 <0.0001  30 vs 10 0.0002 0.0407 30 vs3 <0.0001  0.0407 10 vs 3 <0.0001  NA ^(a)See legend to Table 7 forinformation on the groups. ^(b)p values <0.05 are in bold face foremphasis.

All of the mice in the huIgG treated group and in the two lowest (10 and3 μg) CAS-024 dose groups were euthanized due to growth of their tumors.In contrast, the majority of tumors in the groups of mice treated with200 or 100 μg of CAS-024 regressed to the point that no palpable tumorwas present. By the end of the study, 11/14 (79%) of the mice in each ofthe two highest dose groups and 5/14 (36%) of the mice in the 30 μg dosegroup remained tumor-free (p<0.0001 and 0.0407, respectively, vs. huIgGgroup).

Thus, CAS-024 exhibited dose-dependent inhibitory effects on the growthof established subcutaneous human tumor (DOHH2) xenografts in SCID mice.The two highest dose regimens (100 or 200 μg per IP injection;cumulative dose of 300 or 600 μg, which corresponds to about 16.7 or 33mg/kg, respectively) had similar inhibitory effects and were the mostefficacious of the regimens tested in terms of inhibiting tumor growth,prolonging survival, and inducing complete tumor regression.

Example 8 Efficacy of CAS-024 and Rituxan® as Single Agents in anEstablished Human Tumor (DOHH2) Xenograft Model in SCID Mice

The objective of this study was to examine the efficacy of CAS-024 andRituxan as single agents in a model of established human tumor (DOHH2)xenografts in SCID mice. As set out above, DOHH2 is a CD20⁺CD37⁺ humanB-lymphoblastoid cell line derived from a patient with follicularlymphoma.

Five million DOHH2 cells were injected subcutaneously into the flank offemale CB-17SCID mice (Harlan, Somerville, N.J.) at 6.5 weeks of age. Onday 8 post-tumor inoculation, palpable tumors were apparent in amajority of the mice. The tumor-bearing mice were sorted into fourgroups (n=15 per group; 3 cages of 5 mice for each group) withequivalent mean tumor volumes. The day of the sort was defined as day 0of the study. Tumor diameters were determined with a pair of calipersand tumor volumes were calculated using the formula:V=½[length×(width)²]₃. The baseline mean tumor volume was 228 mm³; themedian baseline tumor size was 227 mm; and the range was 181 to 272 mm³.Mice (15 per treatment group) were treated on days 0, 4, and 8 via IPinjection of 0.2 mL of PBS containing 200 μg human IgG, CAS-024, orRituxan® (for a total of 600 μg after the three treatments). For thehuIgG, CAS-024, and Rituxan® IP treated groups, solutions were preparedin similar volumes and the contents of the tubes were noted on removablelabels. An investigator who was not treating or assessing the miceplaced a color code on each tube and noted the code and identity of thetube contents in a laboratory notebook.

Mice were monitored daily by visual inspection. Weights were determinedweekly, and tumor diameters were determined at least 3 times per week(M, W, F) by an observer blinded (see above) to the treatment groups.Tumor volumes were calculated as described above. Tumor volumes on thelast day that all mice were alive in each group were also expressed interms of tumor volumes relative to day 0, using the formula:

Relative tumor volume on day of interest=(volume on day ofinterest−volume on day 0)volume on day 0

Mice were euthanized if their tumor volume reached more than 1500 mm3(or 1200 mm3 on Fridays). Death is not an endpoint in our tumorprotocols, and unless noted otherwise, “survival” of a mouse wasdetermined by the time it was euthanized due to its tumor volumereaching the predetermined limits. (Our protocol calls for mice to beeuthanized if their tumor volume exceeds the parameters noted above,ulceration of a tumor occurs, the tumor inhibits the mobility of themouse, or if weight loss exceeds 20%.)

All statistical analyses were performed using GraphPad Prism software.Significant differences in mean tumor volumes and mean relative tumorvolumes were determined using a one-way ANOVA for nonparametric data(Kruskal-Wallis test) with Dunn's multiple comparison post test.Significant differences in survival of mice over time were determinedusing Kaplan-Meier survival analysis with a log-rank test for comparingsurvival curves. Significant differences in the incidence of tumor-freemice were determined using Fisher's exact test (p values<0.05 wereconsidered significant).

Mice were euthanized when their tumor volume reached the limitsdescribed above. One mouse in the CAS-024 treatment group was euthanizedon day 45 due to weight loss>20%. This mouse had no apparent SC tumor atthat time, and was treated as censored data for the survival analysis(not euthanized as of day 45 due to tumor growth) and was not includedin the comparison of tumor-free incidence at the end of the study. Noother mice were found dead and none were euthanized due to weight loss,tumor ulceration, or impaired mobility. No overt signs of toxicity orweight loss were observed in any of the treatment groups (data notshown).

The CAS-024 and Rituxan treated mice exhibited a rapid response totreatment. Mean tumor volumes of the CAS-024- and Rituxan®-treatedgroups were significantly lower than that of the human IgG treated groupas early as day 4 (after a single injection of drug) and remained lowerthrough day 11. There were no significant differences in mean tumorvolumes or mean relative tumor volumes between the CAS-024 and Rituxan®treated groups through day 11. The huIgG treated mice were euthanizedstarting on day 11; therefore, comparisons of tumor volumes were notperformed for later time points.

The tumors in the mice treated with huIgG grew rapidly and all mice inthis group were euthanized by day 15. In contrast, by day 15, themajority of tumors in the CAS-024 and Rituxan treated groups hadregressed to the point that no palpable tumor was present. Notably, theresponse to treatment was durable only in the CAS-024 treated group. Bythe end of the study, all of the Rituxan-treated mice were euthanizeddue to growth of their tumors, whereas 10/14 (71%) of the mice in theCAS-024 treated group remained tumor-free. See Table 9. Thus, at the endof the study, the survival curves and the incidence of tumor-free micein the CAS-024 treated group differed significantly from the huIgGcontrol group and the Rituxan® treated group. FIG. 6 shows that CAS-024was statistically superior to Rituxan in the in vivo treatment of thisanimal model of follicular lymphoma.

TABLE 11 Median Survival Time and Incidence of Tumor-Free Mice Fischer'sTreatment p Value Death (other Exact Test Days and Median from Log thanfor Tumor- (Comparison Treatment Cumulative Survival Rank Tumor SizeFree Mice of tumor-free Group Dose Time (Days)^(a) Test^(b) Sacrifice)at Day 81^(c) incidence)^(b) HuIgG Days 0, 4, 8 13 — 0/15  0/15 (0%) NA600 μg CAS-024 Days 0, 4, 8 Undefined ^(d, e) <0.0001 1/15^(f) 10/14(71%) ^(f) <0.0001 IP 600 μg Rituxan ® Days 0, 4, 8 43 <0.0001 0/15 0/15 (0%) NA IP 600 μg ^(a)“Survival” was determined by the day a mousewas euthanized due to tumor growth. Other than one mouse in the CAS-024dose group (see (f)), no mice were euthanized for reasons other thantumor volume reaching the predetermined limit. ^(b)Each group wascompared with the HuIgG treated control group. ^(c)“Tumor-free” mice hadno palpable SC tumors; confirmation of tumor cells absence was notconfirmed by histology. ^(d)The median survival time is undefinedwhen >50% of the mice are alive at the end of the observation period.^(e)Bold-faced values are significantly different from those of HuIgGcontrol. ^(f)One mouse was euthanized on day 45 due to >20% weight loss.The mouse had no apparent SC tumor at that time and was excluded fromthe group for the comparison of tumor-free mice at day 81.

In conclusion, CAS-024 and Rituxan were efficacious as single agents ina human tumor (DOHH2) xenograft model in SCID mice. While both agentscaused an initial tumor regression in the majority of mice, long-termtumor regression was observed only in the group of mice treated withCAS-024 as tumors relapsed after optimal anti-CD20 treatment.Consequently, CAS-024, a humanized anti-CD37 SMIP, shows significantefficacy in pre-clinical tumor xenograft models including models thatshow that Rituxan® treatment fails over time. These results thereforesuggest that CAS-024 treatment of B cell lymphoma and leukemia patientsis beneficial and is a viable alternative treatment in patients who failRituxan® treatment.

Example 9 In Vitro Evaluation of CAS-024 Combined with ChemotherapeuticAgents

It was previously demonstrated that CAS-006 acts synergistically incombination with the chemotherapeutic agent fludarabine to kill chroniclymphocytic leukemia (CLL) cells in vitro (see, e.g., US PatentApplication Publication No. 2007/0059306). As CLL cells do not activelydivide in cell culture in vitro, the data indicate that cellproliferation is not required for the pro-apoptotic effect of CAS-006 orCAS-024 for its synergy with chemotherapeutic agents. The purpose ofthis study, therefore, was to determine whether CAS-024 and variouschemotherapeutic agents were effective on a mantle cell lymphoma (MCL)cell line, Rec-1, that actively grows and divides in cell culture invitro and whether the combination of CAS-024 and a chemotherapeuticagent (drug) would desensitize or enhance the response of mantle celllymphoma cells to various chemotherapeutic agents. The chemotherapeuticagents tested were doxorubicin, vincristine, and fludarabine, which areused to treat non-Hodgkin's lymphoma and other lymphoid malignancies.

Rec-1 cells, a CD37+ human B cell line established from a patient withmantle cell lymphoma, were tested for growth inhibition in response tocrosslinked CAS-024 in the presence or absence of doxorubicin,vincristine, or fludarabine (see FIG. 7). CAS-024 was preincubated withanti-human IgG F(ab)′₂ to crosslink the protein. Cells were culturedwith medium alone or with medium containing various concentrations ofthe crosslinked CAS-024 protein, in the presence or absence of variousconcentrations of doxorubicin, vincristine, or fludarabine. Cultureswere incubated for 96 hours and growth inhibition was assessed using anATP viable cell detection system (i.e., viable cells quantified by ATPrelease).

The Median Effect/Combination Index (CI) method of Chou and Talalay(Adv. Enzyme Regul. 22:27, 1984) was used for data analysis. A numericalvalue, assigned to each drug combination at predefined dose levels,enabled quantitative drug/drug interaction comparisons between differentdrug combinations. Results were expressed as combination indices (CI)vs. effect level, in which effect level represented percent inhibitionof cell growth. The mean CI±SEM for each effect level was averaged overthree experiments. A CI<1.0 was considered synergy, CI=1.0 additivity,and CI>1.0 antagonism. Values presented are the mean±SEM for each effectlevel, averaging three independent assays.

The combination of CAS-024 with vincristine or fludarabine wassynergistic (CI<1.0) and the combination of CAS-024 and doxorubicin wasadditive (CI not significantly different from 1.0). None of CAS-024 andchemotherapeutic agent combinations were antagonistic (CI>1.0) acrossall effect levels. Therefore, the combination of CAS-024 with each ofthe three chemotherapeutic agents tested did not desensitize targetcells to drug-induced growth inhibition, but instead resulted insynergistic or additive inhibitory effects on target cell growth. Apreferred embodiment would be the combination of CAS-024 (SEQ ID NO:253)with vincristine or fludarabine. These data indicate that the efficacyof established chemotherapeutics increase when used in combination withCAS-024.

Example 10 Preliminary Clinical Phase 1/2 Results

As provided herein, pre-clinical studies have demonstrated that CD37SMIP molecules mediate significantly greater direct and natural killer(NK)-cell mediated killing of chronic lymphocytic leukemia (CLL) cellsas compared to other therapeutic antibodies used in CLL. Hence, a Phase1/2, open label, dose escalation study has been initiated in patientswith relapsed chronic lymphocytic leukemia (CLL).

Patients with relapsed/refractory CLL or small lymphocytic lymphoma(SLL) who had adequate organ function, platelets>30,000/mm³ wereeligible. Six doses and two different schedules (cohorts 1-10) have/orwill be studied. The planned doses range from 0.03 mg/kg to 10 mg/kg IVonce a week for 4 doses (cohort 1-6 and 9). The second schedule (cohort7, 8, and 10) will test 3.0, 6.0, or 10.0 mg/kg on days 1, 3 and 5 thefirst week followed by 3 weekly doses. Dose escalation and de-escalationis based on Common Toxicity Criteria Adverse Events (CTC AE) toxicitygrades. Patients may receive 2 additional cycles, if positive biologiceffect after first cycle.

Results: To date, 22 patients have been enrolled (cohort 1-7 and 9) andcompleted treatment (all have received prior fludarabine and rituximabtreatment). Six patients have entered a second cycle and two patientshave entered a third cycle. The patients being treated have gone througha number of prior regimens (e.g., Cohort 4 patients had from 6 to 10(median 6) and Cohort 5 had 5 to 13 (median 9.5) prior regimens). Eightof the ten have high risk genomic features [del(17p13.1), n=5 anddel(11q22.3), n=3]. No dose limiting toxicities or serious adverseevents have occurred. Mild (grade 1-2) infusion toxicity has beenobserved in three patients. Beginning with the 0.3 mg/kg dose, all eightpatients demonstrated evidence of biological activity including patientswith del(17p13.1). Two patients had partial clearing of leukemia cutis,and the median reduction in peripheral lymphocyte count has been 64%(see FIG. 5). One patient had a 99% reduction in peripheral lymphocytecount with no serious adverse events and a continuing response after 3months of treatment (see FIG. 6). One patient had an increase inhemoglobin of 40% and a reduction in lymph node size of 36% asdetermined by CT scan and continues to respond after 3 months oftreatment (see FIG. 7). Two patients had a significant increase inplatelet count.

Conclusion: To date, this CD37 SMIP molecule is a well toleratedtreatment with minimal infusional toxicity and no observed dose limitingtoxicity. There also seems to be any complement involvement sincepatients with severe drops in lymphocyte counts are not showing signs oftumor lysis syndrome. Encouraging reduction in tumor lymphocyte bloodcounts, reduction in lymph node/spleen size, clearing of leukemia cutis,and/or partial clearing of marrow disease, and/or improvement in normalhematopoeitic function in patients with high risk genomic CLL havealready been observed at low, non-saturating doses of CD37 SMIPmolecule.

Example 11 In Vitro Efficacy of CAS024 Combined with Bendamustine

This study was to determine the effects of CAS024, bendamustine, and thecombination of CAS024 and bendamustine on Rec-1 (a mantle cell lymphomacell line) and SU-DHL-6 (a diffuse large cell lymphoma line) cells.

The following human cell lines expressing CD37 were used: Rec-1 andSU-DHL-6 (both from DSMZ, Braunschweig, Germany). Bendamustine(TREANDA®) was purchased from the University of Washington Pharmacy(Seattle, Wash.) and was dissolved in PBS and stored at −20° C. untiluse.

Rec-1 and SU-DHL-6 cells were plated at 1×10⁴ cells/well in 100 μLmedium in 96 well black-sided, black-bottomed plates. Cells were treatedwith various concentrations of CAS024 that had been preincubated withanti-human IgG F(ab)′₂ and plates were incubated for 96 hr at 37° C., 5%CO₂ in the presence of serial dilutions of bendamustine. The finalvolume in each well was 150 μL. After incubation, plates were cooled toroom temperature and labeled with 100 μL/well of ATPlite detectionreagent (Perkin Elmer, Boston, Mass.). The assay measures cellular ATPas a marker for viable cells. Samples were analyzed by detection ofluminescence using a Topcount NXT (Perkin Elmer, Waltham, Mass.) platereader. Data were reduced using a 4-parameter curve fit in Prism(version 4.0, Graphpad Software, San Diego, Calif.) and the IC₅₀ definedas the concentration resulting in 50% inhibition compared to untreatedcultures.

For synergy determination the Median Effect/Combination Index (CI)method was used for data analysis (Chou and Talalay). A numerical value,assigned to each drug combination at predefined dose levels, enablesquantitative drug/drug interaction comparisons between different drugcombinations. The CI values assign interactions into three categories:synergism, additivity, and antagonism (CI<1.0, =1, or >1.0respectively). After labeling and data reduction, Combination Index (CI)values were determined using the Calcusyn software package (Biosoft,Cambridge, UK). The results of two separate experiments show that thecombination of CAS024 with bendamustine resulted in synergisticinhibitory effects on target cell growth (see, FIG. 11). Similar resultswere obtained showing that the combination of CAS024 with bendamustinealso synergistically inhibited SU-DHL-6 cell growth.

Combination effects of CAS-024 with another alkylating agent,chlorambucil, were also determined using the method described above andthe concentrations shown in FIG. 12. Unlike bendamustine, chlorambucilin combination with CAS-024 did not result in synergistic inhibitoryeffects on SU-DHL-6 cell growth (see, FIG. 13)

Example 12 Efficacy of CAS024 Combined with Bendamustine in Human TumorXenograft Model

This study was to compare the efficacy of CAS024 combined withbendamustine against each agent individually administered againstsubcutaneous DOHH2 human tumor xenografts in SCID mice.

Establishment of tumor xenografts and sorting into treatment groups. Asdescribed above, DOHH2 is a CD20⁺CD37⁺ human B-lymphoblastoid cell linederived from a patient with follicular lymphoma. Five million DOHH2cells were injected subcutaneously into the flank of female CB-17 SCIDmice. On day 8 post-tumor inoculation, palpable tumors were apparent inmajority of mice. The tumor-bearing mice were sorted into five groupswith equivalent mean tumor volumes (n=15 per group; 3 cages of 5 micefor each group). The day of the sort was defined as day 0. Tumordiameters were determined with a pair of calipers and tumor volumes werecalculated using the formula: V=½[length×(width)²]. The baseline meantumor volume was 231 mm³, the median baseline tumor size was 229 mm³,and the range was 201 to 261 mm³.

In vivo treatment. Groups of mice were treated with an injection of 0.2mL of PBS containing 10 μg huIgG (days 0, 4, 8 IV), 10 μg CAS024 (days0, 4, 8 IV), 10 mg/kg Bendamustine (0, 2, 4, 7, 9 IP), or 10 μg CAS024(days 0, 4, 8 IV) AND 10 mg/kg Bendamustine (0, 2, 4, 7, 9 IP).

Monitoring and endpoints. Mice were monitored daily by visualinspection. Weights were determined weekly, and tumor diameters weredetermined at least 3 times per week (M, W, F) by an observer blinded(see above) to the treatment groups. Tumor volumes were calculated asdescribed above.

Mice were euthanized if their tumor volume reached more than 1500 mm³(or 1200 mm³ on Fridays). Death was not an endpoint in this study, andunless noted otherwise, “survival” of a mouse was determined by the timeit was euthanized due to its tumor volume reaching the predeterminedlimits. Mice were euthanized if their tumor volume exceeded theparameters noted above, ulceration of a tumor occurs, the tumor inhibitsthe mobility of the mouse, or if weight loss exceeds 20%.

Statistical analyses. All statistical analyses were performed usingGraphPad Prism software. Significant differences in mean tumor volumesand mean relative tumor volumes were determined using a one-way ANOVAfor nonparametric data (Kruskal-Wallis test) with Dunn's multiplecomparison post test. Significant differences in survival of mice overtime were determined using Kaplan-Meier survival analysis with alog-rank test for comparing survival curves. Significant differences inthe incidence of tumor-free mice were determined using Fisher's exacttest. p values<0.05 were considered significant.

In the Bendamustine treated groups scruffy coats and diarrhea were seenstarting around day 6. On day 10, one mouse in the CAS024+ Bendamustinetreatment group was euthanized due to ≧20% weight loss. This mouse wastreated as censored data for the analysis of survival curves. Noclinical signs of toxicity were seen in the CAS024 alone treatmentgroup.

All treatments, demonstrated an inhibitory effect on the growth of DOHH2compared to huIgG. On day 13 (which was the last day all mice werealive) the mean tumor volume and mean relative tumor volume of all thetreatment groups were statistically different than the huIgG controlgroup of mice (FIGS. 14A and 4B). A significant difference in mean tumorvolumes and mean relative tumor volumes was also seen betweenBendamustine and the CAS024+ Bendamustine combination treatment group.There were no significant differences in mean tumor volumes or meanrelative tumor volumes between any two other treatment groups. Meantumor volumes over time of the four groups are shown in FIG. 15.

The tumors in the mice treated with huIgG grew rapidly, and all of themice in this group were euthanized by day 17. As shown in FIG. 16 andsummarized in Tables 12 and 13, the survival of mice dosed with any ofthe treatment groups was prolonged compared to the huIgG treated group(p≦0.0001 for all groups). There was also a significant differencebetween the survival curves of all three treatment groups and each otherwith the CAS024/bendamustine combination being superior to either singleagent.

None of the huIgG-treated mice were alive (thus none were tumor-free) atthe end of the study (day 34) (FIG. 17 and Table 12). The incidence oftumor-free mice in the other groups was 0/15 (0%) in the CAS024 andBendamustine treatment groups and 2/14 (14%) in the CAS024+Bendamustinecombination treatment group. There was no significant difference in theincidence of tumor-free mice between any of the treatment groups.

TABLE 12 Median Survival Time and Incidence of Tumor-Free Mice at theend of the Observation Period Median Tumor-Free Treatment Survival TimeIncidence at Group^(a) Treatment Days (Days)^(a) End of Study huIgG Days0, 4, 8 15 0/15 (0%) CAS024 Days 0, 4, 8   17 ^(b) 0/15 (0%) 10 μgBendamustine Days 0, 2, 4, 7, 9 17 0/15 (0%) 10 mg/kg CAS024 + Days 0,4, 8 24   2/14 (14%)^(d) Bendamustine 0, 2, 4, 7, 9 ^(a)“Survival” of amouse was determined by the day it was euthanized due to tumor growth.One mouse in the CAS024 + Bendamustine combo group was euthanized on day10 due to ≧20% weight loss. This mouse was treated as censored data whencalculating survival curves. No other mice were euthanized for reasonsother than its tumor volume reaching the predetermined limit. ^(b)Valuesin bold face indicate that the survival curves of the indicated groupare significantly different from those of huIgG control (p < 0.0001 forall treatment groups; log rank test). ^(c)“Tumor-free” mice have nopalpable SC tumors. The absence of tumor cells was not confirmed byhistology. Study ended on day 34. ^(d)In the CAS024 + Bendamustine combogroup one mouse was euthanized on day 10 due to ≧20% weight loss. Noother mice were euthanized for toxicity reasons.

TABLE 13 p Values for Comparison of Survival Curves Between TreatedGroups p Values for Comparison of survival curves (log-rank test)TRU-016 + huIgG TRU-016 Bendamustine Bendamustine huIgG NA <0.0001<0.0001 <0.0001 TRU-016 <0.0001^(a) NA 0.0050 0.01 Bendamustine <0.00010.0050 NA <0.0001 TRU-016 + <0.0001 0.01 <0.0001 NA Bendamustine

This study shows that CAS024 combined with Bendamustine exhibitedinhibitory effects on the growth of DOHH2 tumors in SCID mice greaterthan that seen with either agent alone.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1.-3. (canceled)
 4. A CD37-specific binding molecule, comprising anamino acid sequence as set forth in SEQ ID NO:253.
 5. The CD37-specificbinding molecule of claim 4, consisting of the amino acid sequence setforth in SEQ ID NO:253. 6.-8. (canceled)
 9. A composition comprising ahumanized CD37-specific binding molecule according to claim 4 and apharmaceutically acceptable carrier. 10.-29. (canceled)
 30. Acomposition comprising a humanized CD37-specific binding moleculeaccording to claim 5 and a pharmaceutically acceptable carrier.